distinct inhibitory effects of dihydroteleocidin b and the ... · l1, is capable of differentiating...

[CANCER RESEARCH 43, 4974-4979, October 1983]

Distinct Inhibitory Effects of Dihydroteleocidin B and the Phorbol

Ester Tumor Promoters on the Adipocyte Differentiation of3T3-L1 Cells1

Yoshiko Shimizu, Nobuyoshi Shimizu,2 Hirota Fujiki, and Takashi Sugimura

Department of Cellular and Developmental Biology, University of Arizona, Tucson, Arizona 85721 [Y. S., N. S.], and National Cancer Center Research Institute 5-1-1Tsukiji, Tokyo, Japan [H. F., T. S.]

ABSTRACT

An indole alkaloid tumor promoter, dihydroteleocidin B, wasable to modulate a membrane property of 3T3-L1 preadipocytes,

showing an almost complete reduction of epidermal growthfactor binding capacity. This receptor modulating potency ofdihydroteleocidin B, was 10 times that of a phorbol ester tumorpromoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Dihydro

teleocidin B, however, had little effect on the epidermal growthfactor receptors of the adipocyte stage of 3T3-L1. Adipocytedifferentiation was induced by treating growth-arrested 3T3-L1cells with dexamethasone and 1-methyl-3-isobutylxanthine for

48 hr. These inducers initiated DNA synthesis, led to one fullcycle of cell division, and triggered the adipocyte differentiationprogram. Dihydroteleocidin B almost completely inhibited thisdifferentiation at concentrations of 1 to 10 ng/ml (10~9 to 10~8

M). The inhibition was observed regardless of when the tumorpromoter was added: before, during, or after the addition ofinducers. Similar inhibition was also observed by TPA, but withover 90% less efficiency than that of dihydroteleocidin B. TPAwas most effective when it was added during the inducer treatment. Both dihydroteleocidin B and TPA stimulated DNA synthesis to the same level during the initial 22 hr. The DNA synthesisstimulated by dihydroteleocidin B resulted in extraordinary enhancement of cell proliferation, whereas TPA-treated 3T3-L1

cells did not divide. These findings suggest that dihydroteleocidinB and TPA have distinct potencies in interfering with the mechanisms of adipocyte differentiation and that presumably they aredifferent in action of tumorigenesis.

INTRODUCTION

There are a number of compounds found in the environmentthat are not themselves carcinogenic but are able to promotestrongly the development of skin tumors in mice pretreated witha subcarcinogenic dose of a chemical carcinogen (1-4, 37, 41).

These agents, tumor promoters, are phorbol ester derivatives,such as TPA3 (16,17), and derivatives of indole alkaloids, such

as DHTB, a newly discovered tumor promoter (12, 13). Tumorpromoters exhibit many different biological and biochemical effects on mouse skin and cells in culture (10,11,44). They appear

1Supported by NIH Grant GM24375 and the US-Japan Cooperative Cancer

Research Program.2To whom requests for reprints should be addressed.3The abbreviations used are: TPA, 12-O-tetradecanoylphorboM 3-acetate;

DHTB, dihydroteleocidin B; DEX, dexamethasone; MIX, 1-methyl-3-isobutylxan-thine; DME, Oulbecco's modified Eagle's medium; EBSS-BSA, Earle's balanced

salt solution containing 5 mw 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidand bovine serum albumin; EGF, epidermal growth factor; PDEH, phorbol-12,13-diester 12-ester hydrolase.

Received October 12,1982; accepted July 13,1983.

to act primarily by modulating gene expression and cell differentiation through their binding to specific cell surface receptors(9, 40).

In addition to studies on the mechanisms of carcinogenesisand tumor promotion, we are interested in utilizing these tumor-promoting compounds as tools for studying the fundamentalmechanisms underlying normal cell differentiation at the molecular level. A variant line of mouse Swiss/3T3 fibroblasts, 3T3-

L1, is capable of differentiating into adipocytes (14, 15). A highfrequency of induction of differentiation can be obtained bytreating growth-arrested 3T3-L1 cells with DEX and MIX (29). In

this report, we compared the effect(s) of DHTB with that of TPAon the adipocyte differentiation of 3T3-L1 cells. There are re

markable differences between DHTB and TPA in their potencyand mode of inhibiting the terminal differentiation of 3T3-L1 cells.

MATERIALS AND METHODS

Chemicals. TPA and its derivatives were purchased from Consolidated Midland Corp. (Brewster, N. Y.). DHTB was prepared by thecatalytic reduction of teleocidin B isolated from Streptomyces medioci-

dicus (12, 39). The compounds were dissolved in dimethyl sulfoxide andstored at -20Â° until use. All other chemicals were purchased from Sigma

Chemical Co. (St. Louis, Mo.) unless otherwise noted.Cell growth. 3T3-L1 cells were purchased from the American Type

Culture Collection (Rockville, Md.). They were routinely grown in DMEmedium supplemented with 10% fetal calf serum, penicillin (100 units/ml), and streptomycin (100 ^g/ml; Grand Island Biological Co., SantaClara, Calif.). Cultures were maintained at 37Â° in a 95% air-5% CO2

atmosphere.Differentiation. 3T3-L1 cells (~1.5 x 105) were plated in 35-mm

dishes in 2 ml of DME medium containing 10% fetal calf serum. Twodays after the cells reached confluency, the medium was replaced withfresh DME medium containing 0.25 /Â¿MDEX and 0.5 mivi MIX. Two dayslater, the inducer-containing medium was replaced with fresh DME

medium. Medium was changed every 3 days thereafter. The drugs wereadded at various times as indicated. For some experiments, insulin wasadded at 1 Â¿/g/mlalone or together with DEX and MIX for induction.Adipocytes were identified by the presence of large lipid droplets whichstain with oil red 0. Cell number was counted in a hemocytometer afterdetaching cells by trypsinization.

DNA Synthesis. Medium was removed from confluent cultures of 3T3-

L1 cells and replaced with fresh DME medium containing 5% fetal calfserum, and incubation was continued for 3 days. [3H]Thymidine was

then added to the conditioned medium to 1 MM (1 /Â¿Ci/ml;ICN, Irvine,Calif.), and the cultures were incubated for 22 hr at 37Â°.The cultures

were placed on ice, the medium was removed, and the cultures werewashed 3 times with Dulbecco's phosphate-buffered saline (2.7 HIM KCI,

137 HIM NaCI, 1.5 muÃKH2PO4, 8 HIM NaaHPCv, 1 mw CaCI2, and 0.5rriM MgCI2) containing 50 rtiM unlabeled thymidine, overlaid with ice-cold10% trichloroacetic acid and kept at 4Â°overnight. The cells were washed

twice with 5% trichloroacetic acid and dissolved in 1 N NaOH. Radioac-

4974 CANCER RESEARCH VOL. 43

on June 18, 2020. © 1983 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

Tumor Promoters andAdipocyte Differentiation

tivity was counted with 3a70b solution (Research International Products;Mount Prospect, III.) in a Packard scintillation spectrometer as before

(34).EGF Binding. Confluent cultures in 60-mm dishes were placed on ice

and with EBSS-BSA (33). One ml of EBSS-BSA buffer was added toeach dish, 125I-EGFwas added to 1 to 2 x 10~10M, and the dishes were

incubated at 23Â°for 60 min. The drugs were added in the cultures 2 hr

before the binding assay. After incubation, the cells were placed on ice,washed with EBSS-BSA, and solubilized in 1 ml of 0.5 N NaOH. Radioactivity was determined in a Beckman 4000 gamma counter. 125I-EGF

used for the present study was produced from receptor grade EGF(Collaborative Research, Waltham, Mass.) by the chloramine-T iodination

method. Nonspecific binding was measured and subtracted from totalbinding as before (33).

RESULTS

One of the striking effects of tumor promoters, such as TPA,on cells in culture is the reduction of 125I-EGF binding to cell

surface receptors (5,20,40). This is not due to direct competitionof TPA with EGF for its receptors but is attributed to themodulation by TPA of the membrane in an as yet unknownfashion (21, 24, 35). In 3T3-L1 cells, the EGF-binding capacity is

constant before and after adipocyte differentiation, in contrast tothe dramatic increase in the insulin-binding capacity after differ

entiation (29). We have tested the effects of DHTB and TPA onthe binding of EGF (Chart 1) and found that both DHTB and TPAsubstantially reduce the EGF-binding capacity of 3T3-L1 cells in

the preadipocyte state and that this receptor down regulation byDHTB is 10 times more efficient than that of TPA (Chart 1-4).EGF binding to the cells which have differentiated to adipocytes(Chart 10) was reduced by TPA to the same extent as that inthe preadipocyte state, but the effect was 10 times less efficientin the adipocytes than in the preadipocytes. The effect of DHTBon the EGF-binding capacity of 3T3-L1 adipocytes was evenless, reducing it by 20% at 100-ng/ml concentrations of DHTB.

These differential modulations of EGF receptors by DHTB andTPA suggest that these tumor promoters may be acting throughdistinct mechanisms; in other words, their membrane-modulatory

effects may be transmitted to EGF receptors in different manners.

The 3T3-L1 cells can be maintained at confluency in medium

containing 10% fetal calf serum for more than 1 week withoutdifferentiation (Chart 2). Differentiation, however, is stimulated

KXH-

"01 10 WO "~ 0.1

TUMOR PROMOTER(ng/ml)

io i5Ã´

Chart 1. Reduction of EGF binding capacity by tumor promoters. 3T3-L1 cells(2 x 10e) were incubated with various concentrations of DHTB or TPA for 2 hr at37Â°,and then with 2 x ICT10 M 125I-EGFin EBSS-BSA buffer for 1 hr at 23Â°.Thecells were washed with EBSS-BSA, and the radioactivity was counted. Average of2 determinations. A, 3T3-L1 cells in the preadipocyte state; B, 3T3-L1 cells in theadipocyte state.

by treating confluent cells for 2 days with the inducers DEX andMIX (29). By the sixth day after inducer treatment, 40 to 70% ofthe cells have converted into adipocytes, which accumulate largelipid droplets in their cytoplasm. We examined the effects ofDHTB and TPA on this differentiation program. Adipocyte differentiation was almost completely inhibited when DHTB was addedto the cultures at 10 ng/ml (Chart 2). The same concentration ofTPA also inhibited adipocyte differentiation but to a much lesserextent. In these experiments, tumor promoters were addedconcurrently with the inducers and kept present for the next 4days in the culture medium (Chart 2, arrows). Partial inhibition ofadipocyte differentiation by TPA is consistent with previous workby Diamond ef a/. (7). Other phorbol esters, such as 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate and phorbol dibutyrate

which are known as weak promoters or nonpromoters, causedno inhibition of adipocyte differentiation (data not shown). Next,we tested whether DHTB exhibits its inhibitory effect whenadded before DEX-MIX treatment, during the inducer treatment,

or after the induction. As seen in Chart 3, DHTB significantlyinhibited adipocyte differentiation no matter when it was added.Addition of DHTB even before or after induction was effective inpreventing differentiation. In the case of TPA (Chart 4), adipocyte

Chart 2. Time course of adipocyte differentiation and its inhibition by tumorpromoters. Confluent 3T3-L1 cells were treated with the inducers DEX and MIXfor 2 days. DHTB or TPA was added at 10 ng/ml concurrently with the inducersand kept present for the next 4 days.

DAYSChart 3. Inhibition of adipocyte differentiation by DHTB under different sched

ules of addition. Adipocyte differentiation was induced by treating confluent 3T3-L1 cells with DEX and MIX for 2 days. DHTB (10 ng/ml) was added for 1 day justbefore induction (A), during the induction period (B), and for 1 day just afterinduction (C).

OCTOBER 1983 4975



Y. Shimizu et al.

differentiation was not inhibited if the drug was added and thenremoved prior to induction. Effective inhibition, although onlypartial, was seen when TPA was added during the inductionperiod. The TPA did not cancel out the postinduction events.The difference in inhibitory ability between DHTB and TPA isillustrated in Chart 5A, where dose dependency of inhibition was

DAYS10

Chart 4. Inhibition of adipocyte differentiation by TPA under different schedulesof addition. Induction of adipocyte differentiation was with DEX and MIX from Day0 to Day 2. TPA (10 ng/ml) was added for 2 days before induction IA), during theinduction period (B), and for 1 day after the induction period (C).

50

~Ã”5 ÃŽ io 180 1Ã›OO


Chart 5. Dose-dependent inhibition of adipocyte differentiation by DHTB andTPA. A, induction of adipocyte differentiation was with DEX and MIX from Day 0to Day 2. Tumor promoters were added for 2 days before induction (O, â€¢),duringthe induction period p, â€¢),for 2 days after the induction period (A, A), orconcurrently with the inducers and replenished every day for 6 days (V, T).Adipocyte differentiation was measured on Day 6. B, adipocyte differentiation wasinduced with DEX, MIX, and insulin (1 ng/ml) for 2 days. Tumor promoters wereadded during the induction period. Adipocyte differentiation was measured on Day6.

tested using 4 different treatment schedules. DHTB at 10 ng/mlconsistently gave almost complete inhibition, while TPA wasinhibitory only when it was present during the induction period.Distinct inhibitory profiles were also obtained under the conditions where adipocyte differentiation was accelerated by addinginsulin together with inducers (Chart SB). Adipocyte differentiation could be induced by insulin (6 //g/ml) alone but at a lowfrequency (1 to 5%). This insulin-induced differentiation was also

inhibited by both DHTB and TPA (data not shown). However, acomparison of potencies was not possible due to the low frequency of differentiation. These results together suggest thatboth DHTB and TPA can truly antagonize the adipocyte differentiation program but that DHTB is apparently a pluripotentagent.

When growth-arrested 3T3-L1 cells were treated with the

inducers DEX and MIX, their DNA synthesis was initiated within22 hr and increased to 2.8 times the control (Table 1). DEX orMIX alone stimulated DNA synthesis only by 50 to 60%. As hasbeen reported (8), TPA stimulated DNA synthesis 1.9 times morethan the control and further enhanced the mitogenic effect of theinducers. Strikingly, DHTB was a 3 times more potent stimulatorfor DNA synthesis and exhibited remarkable synergistic stimulation with the inducers. Chart 6 illustrates the dose-dependentmanner of DNA synthesis stimulation in 3T3-L1 cells and again

shows the difference in potency between DHTB and TPA. Themaximum levels of DNA synthesis during 22-hr treatment werealmost the same. As seen in Chart 7, 3T3-L1 cells which re

sponded to the inducers, DEX and MIX, underwent cell division(compare cell numbers at zero tumor promoters). This inducer-

Table 1Effects of tumor promoters and differentiation inducers on DNA synthesis in 3T3-

L1 cells

AdditionDNA synthesized (pHJthymi-

dine, cpm x 10^22 hr) Ratio to controlNone(control)8DEX,

0.25MMMIX,0.5niMDEX+MIXTPA,10ng/mlTPA

+ DEX +MIXDHTB,10ng/mlDHTB

+ DEX + MIX43.2

Â±3.3*65.4

Â±2.567.2Â±3.3121.3Â±

7.080.8Â±3.2284.4Â±29.4276.1Â±12.6490.8Â± 8.61.001.511.562.811.876.586.3911.4

Basal level of DNA synthesis occurring in confluent cells. Average of 2determinations.

6 Mean Â±S.E.

Dihydro-teleocidinB

1 10 100 1000TUMOR PROMOTER

(ng/ml)Charte. Dose-dependent stimulation of DNA synthesis by tumor promoters.

Confluent 3T3-L1 cells were treated with the indicated concentrations of DHTB orTPA. Incorporation of [3H]thymidine was measured for 22 hr from the time of drug

addition.




Tumor Promoters andAdipocyte Differentiation

Vf86

.1l ~Oi 1 10 105 Ã•OO


Chart?. Effects of DHTB and TPA on 3T3-L1 cell proliferation. Drugs wereadded at the indicated concentrations 2 days after confluency and kept for 2 days.Ind.. inducers (DEX and MIX); â€¢.inducers only; D, no addition. Cell number wascounted on Day 6.

mediated cell division is thought to be a necessary step fortriggering the adipocyte differentiation program (23, 29). Treatment with DHTB apparently stimulated cell proliferation andincreased cell number to twice the inducer-stimulated level, while

TPA had little effect on cell division. Thus, inhibition of adipocytedifferentiation by DHTB is attributed to an overwhelming cellgrowth while the inhibition of TPA is not. This notion was furthersupported by the observations shown in Fig. 1. Gross morphology changes and/or increase of cell density is seen when 3T3-

L1 cells were treated with DHTB (10 ng/ml) regardless of theinducer treatments (Fig. 1, C, D, and G). TPA had no such effectson 3T3-L1 cells even at 100 ng/ml (Fig. 1, B, F, and H).

Thus, the indole alkaloid tumor promoter DHTB is a highlypotent inhibitor of adipocyte differentiation. The inhibition appears to be due to unscheduled stimulation of DNA synthesis.The detailed profile of the inhibition by DHTB is different fromthat of TPA in several aspects.

DISCUSSION

We have described in this paper that the indole alkaloid tumorpromoter DHTB is a significantly more potent inhibitor for adipocyte differentiation of 3T3-L1 cells than is the phorbol ester

tumor promoter TPA. We have found that addition of DHTB orTPA to 3T3-L1 cells, which had grown to confluency and quies

cence, strongly stimulates the cells to initiate DNA synthesis butthat only DHTB facilitated cell division. TPA did not trigger themitosis of 3T3-L1 cells.

Recent studies have shown that both TPA and DHTB stimulategrowth-arrested mouse fibroblastic cells to initiate DNA synthesis

and undergo several rounds of cell division (26). These fibroblastic cells include the Swiss/3T3 line from which the 3T3-L1

preadipocyte line was established (15). It is also known that TPAalone is not mitogenic for these cells in serum-free medium but

rather that it acts synergistically with numerous growth factors(8,26). In the above-mentioned TPA-responsive fibroblastic cells,

it was observed that an increase in cell number is correlated withgross morphological changes (26); while in the TPA-treated 3T3-

L1 cells, there was no obvious morphological change. There arevarious observations in cells other than fibroblasts that TPAinhibits differentiation in certain cell systems whereas it inducesdifferentiation in other systems (12, 13, 19, 27). DHTB also has

reciprocal effects (13). What sort of cell physiology regulates thedirection of these effects is at present not known.

It is believed that TPA and DHTB have equal potencies in theinduction of various biochemical changes (12, 13, 19, 27, 31,40). There is one exception (18) in that DHTB is 100 times moreeffective than TPA in enhancing methylcholanthrene-inducedmalignant transformation of A31-1-1 cells. The results found in3T3-L1 cells provide an additional case in which the potency of

DHTB is shown to be different from that of TPA with respect toinhibition of differentiation.

Previously, Diamond et al. (7) reported that TPA inhibits thespontaneous adipocyte differentiation of clone A31-T of 3T3fibroblasts at concentrations of 10"8 to 10~7 M (10 to 100 ng/

ml). Under their conditions, however, only 5 to 6% of the cellpopulation differentiated to mature adipocytes, and differentiation took 2 weeks after confluency. We have used 3T3-L1

preadipocytes which are able to differentiate into adipocytesunder chemically defined induction conditions. The 3T3-L1 cells

differentiate at a frequency of 40 to 70% within 1 week. Ourobservations using the 3T3-L1 system not only confirmed theTPA-mediated inhibition of adipocyte differentiation observed by

Diamond eÃal. (7) but also provided the basis for further analysisof its action. TPA was effective only when added during theinduction period. In contrast, DHTB caused almost completeinhibition of differentiation regardless of the timing of its additionrelative to the induction period. Thus, DHTB appears to act atmultiple physiological states of 3T3-L1 cells.

Our present work and others provided evidence which supports the following view on the adipocyte differentiation program.3T3-L1 cells, the growth of which is arrested at the G0-Gi stage

of the cell cycle, respond to the inducers, initiate DNA synthesis,and divide (Gs). The divided daughter cells appear to be under aregulatory mechanism in which the cells become arrested inanother state, GD. From this state they proceed to differentiateinto adipocytes. The designation of these states has been discussed previously (23, 32).

Unlike TPA, DHTB forced the growth-arrested 3T3-L1 cells to

divide. DHTB, when added before the induction period, stimulated several rounds of cell division, and this excess cell proliferation did not allow the cells to enter into GD to trigger thedifferentiation program. The effect of DHTB added during theinduction period could be accounted for by a mechanism in whichcompetition with the action of the inducers may be involved. Themechanism by which the inducers MIX and DEX facilitate adipocyte differentiation of 3T3-L1 cells is not fully understood (29).

It is, however, known that changes in prostaglandin metabolismin concert with the cyclic adenosine 3':5'-monophosphate level

are important early cellular responses to the inducers (6,14, 22,25, 26, 29-31, 42, 43, 46). The inhibitory action of TPA was

observed only during this period and could be due to the samemechanism. In other words, both tumor promoters efficientlyblock the entry of the cell into the GDstate, inhibiting expressionof the genes involved in adipocyte differentiation. DHTB addedafter the induction period appears to act on the cells at the GDstate and may interfere with the efficient production and functionof various enzymes related to adipocyte differentiation, namely,the expression and maintenance of the adipocyte phenotype inthe committed GD state cells are inhibited. It has been reportedthat TPA binds to specific surface receptors and that its interaction leads to an increase in membrane fluidity resulting in the

OCTOBER 1983 4977



Y. Shimizu et al.

modulation of certain membrane functions (9, 10, 45). It is alsoreported that DHTB inhibits the specific binding of a TPA analogue, phorbal dibutyrate, to its receptors (40). Modulation ofone cell membrane property, which was measured as a reductionof 125I-EGF binding to cell surface receptors, was greater by

DHTB than by TPA when the cells were in the preadipocytestate. The inverse relationship was observed when cells were inthe adipocyte state. The influence of DHTB on membrane fluiditymay be restricted by the high content of cellular lipids. Theseobservations suggest that membrane modulation as a postreceptor event is a reflection of more complex phenomena thanpreviously appreciated.

Recently, Shoyab ef al. (36) have found the enzyme PDEH inmouse and human liver. This enzyme converts biologically activephorbol-12,13-diesters to the inactive phorbol-13-monoester.

That the weak inhibitory activity of TPA is regulated by the levelof PDEH activity in 3T3-L1 cells is unlikely since replenishment

of TPA in the medium did not increase the inhibition to the levelof DHTB. Enzymes equivalent to PDEH have not been reportedfor DHTB.

Recently, we have observed that nicotinamide and some of itsanalogues inhibit adipocyte differentiation of 3T3-L1 cells (23).This inhibition was correlated with the prevention of inducer-

stimulated cell growth. In other words, nicotinamide inhibiteddifferentiation by preventing 3T3-L1 cells from entering the dif

ferentiation program. Together with other evidence, we supported the previous proposition (28) that adipocyte differentiationinvolves ADP ribosylation, the proposed role of which resides inregulating activity of chromatin structure and function (38). It willbe interesting to examine whether DHTB affects the activity ofpoly(adenosine diphosphoribose) synthetase and/or modificationof chromatin function through ADP ribosylation in 3T3-L1 cells.

The actions of DHTB and TPA are largely similar. However, inthis study, it was shown that there are significant differences inthe prevention of adipocyte differentiation. These 2 tumor promoters may, therefore, serve as agents to further compare anddissect the mechanisms of adipocyte differentiation as well asthe mode of action of tumor promoters at the molecular level.

REFERENCES

1. Baird, W. M., and Boutwell, R. K. Tumor promoting activity of phorbol and fourdiesters of phorbol in mouse skin. Cancer Res., 37:1974-1079, 1971.

2. BerentHum, I. The cocarcinogenic action of croton resin. Cancer Res., 7: 44-48,1941.

3. Berenblum. I. Established principles and unresolved problems in carcinogen-esis. J. Nati. Cancer Inst, 60: 723-726,1978.

4. Boutwell, R. K. Some biological aspects of skin carcinogenesis. Prog. Exp.Tumor. Res. Mol. Biol., 4: 207-250, 1974.

5. Brown, K. D., Dicker, P., and Rozengurt, E. Inhibition of epidermal growthfactor binding to surface receptors by tumor promoters. Biochem. Biophys.Res. Commun., 86:1037-1043, 1979.

6. Chang, T. H., Williams, I., and Pdakis, E. Differentiation of 3T3-L1 fibroblaststo adipocytes: loss of stimulation of uridine and 2-deoxyglucose transport byPGF 2 in the course of differentiation of the 3T3-L1 cell line. Exp. Cell Res.,720:395-403, 1979.

7. Diamond, L, O'Brien, T. G., and Rovera, G. Inhibiton of adipose conversion of

3T3 fibroblasts by tumour promoters. Nature (Lond.) 269: 247-248,1977.8. Dicker, P., and Rozengurt, E. Synergistic stimulation of early events and DNA

synthesis by phorbol esters, polypeptide growth factors, and retinoids incultured fibroblasts. J. Supramol. Struct., 77: 79-93,1979.

9. Driedger, P. E., and Blumberg, P. M. Specific binding of phorbol ester tumorpromoters. Proc. Nati. Acad. Sei. U. S. A., 77: 567-571, 1980.

10. Fisher. P. B., Flamm, M., Schachter, D., and Weinstein, l. B. Tumor promotersproduce membrane changes detected by fluorescence polarization. Biochem.Biophys. Res. Commun., 86:1063-1067,1979.

11. Fisher, P. B., Miranda, A. F., Mufson, R. A., Weinstein, L. S., Fujiki, H.,

Sugimura, T., and Weinstein, l. B. Effects of teleocidin and the phorbol estertumor promoters on cell transformation, differentiation, and phospholipid metabolism. Cancer Res., 42: 2829-2835,1982.

12. Fujiki, H., Mori, M., Nakayasu, M., Terada, M., and Sugimura, T. A possiblenaturally occurring tumor promoter, teleocidin B from Streptomyces. Biochem.Biophys. Res. Commun., 90: 976-983,1979.

13. Fujiki, H., Mori, M., Nakayasu, M., Terada, M., Sugimura, T., and Moore, R.E. Indole alkaloids; dihydroteleocidin B, teleocidin and lyngbyatoxin A, asmembers of a new class of tumor promoters. Proc. Nati. Acad. Sei. U. S. A.,78:3872-3876,1981.

14. Furstenberger, G., and Marks, P. Indomethacin inhibition of cell proliferationby the phorbol ester TPA is reversed by prostaglandin E2 in mouse epidermisin vivo. Biochem. Biophys. Res. Commun., 84:1103-1107,1978.

15. Green, H., and Kehinde, O. Spontaneous heritable changes leading to increased adipose conversion in 3T3 cells. Cell, 7: 105-113,1976.

16. Hecker, E. Phorbol esters from croton oilâ€”chemical nature and biologicalactivities. Naturwissenschaften, 54: 282-284, 1967.

17. Hecker, E. Isolation and characterization of the carcinogenic principles fromcroton oil. Methods Cancer Res., 6: 439-484,1971.

18. Hirakawa, T., Kakunaga, T., Fujiki, H., and Sugimura, T. A new tumor-promoting agent, dihydroteleocidin B, markedly enhances chemically inducedmalignant cell transformation. Science (Wash. D. C.), 276: 527-529,1982.

19. Hoshino, H., Miwa, M., Fujiki, H., and Sugimura, T. Aggregation of humanlyrnphoblastoid cells by tumor-promoting phorbol esters and dihydroteleocinB. Biochem. Biophys. Res. Commun., 95: 842-848,1980.

20. Lee, L.-S., and Weinstein, I. B. Tumor-promoting phorbol esters inhibit bindingof epidermal growth factor to cellular receptors. Science (Wash. D. C.), 202:313-315,1978.

21. Lee, L.-S., and Weinstein, I. B. Mechanism of tumor promoter inhibition ofcellular binding of epidermal growth factor. Proc. Nati. Acad. SÃ¤.U. S. A., 76:5168-5172,1979.

22. Levine, L., and Hassid, A. Effects of phorbol-12, 13-diesters on prostaglandinproduction and phospholipase activity in canine kidney (MDCK) cells. Biochem.Biophys. Res. Commun., 79: 477-483, 1977.

23. Lewis, J. E., Shimizu, Y., and Shimizu, N. Nicotinamide inhibits adipocytedifferentiation of 3T3-L1 cells. FEBS Lett., Ã•46:37-41,1982.

24. Magun, B. E., Matrisian, L. M., and Bowden, G. T. Epidermal growth factor:ability of tumor promoters to alter its degradation, receptor affinity and receptornumber. J. Btot. Chem., 255: 6373-6381,1980.

25. Mufson, R. A., Simsiman, R., and Boutwell, R. K. Increased 3':5'cydic

adenosine monophosphate phosphodiesterase activity in the epidermis ofphorbol ester treated mouse skin and in papillomas. Cancer Res., 39: 2036-

2041, 1979.26. Mufson, R. A., and Weinstein, I. B. Phorbol ester tumor promoters modulate

cellular differentiation. In: M. E. Buckingham and M. J. Clemens (eds.), Biochemistry of Cellular Regulation, Vol. 3, pp. 179-196. Boca Raton, Fla.: CRC

Press, Inc., 1981.27. Nakayasu, M., Fujiki, H., Mori, M., Sugimura, T., and Moore, R. D. Teleocidin,

lyngbyatoxin A and their hydrogenated derivatives, possible tumor promoters,induce terminal differentiation in HL-60 cells. Cancer Lett., 72:271-277,1981.

28. Pekala, P. H., Lane, M. D. Watkins, P. A., and Moss, J. On the mechanism ofpreadipocyte differentiation: masking of poly (ADP-ribose) synthetase activityduring differentiation of 3T3-L1 preadipocytes. J. Biol. Chem., 256: 4871-

4876,1981.29. Rubin, C. S., Hirsch, A., Fung, C., and Rosen, O. M. Development of hormone

receptors and hormonal responsiveness in vitro: insulin receptors and insulinsensitivity in the preadipocyte and adipocyte forms of 3T3-L1 cells. J. Biol.Chem., 253: 7570-7578,1978.

30. Russell, T. R., and Ho, R.-J. Conversion of 3T3 fibroblasts into adipose cells:triggering of differentiation by prostaglandin F2 and 1-methyl-3-isobutylxan-thine. Proc. Nati. Acad. Sei. U. S. A., 73: 4516-4520,1976.

31. Sakamoto, H., Terada, M., Fujiki, H., Mori, M., Nakayasu, M., Sugimura, T.,and Weinstein, I. B. Stimulation of prostaglandin production and cholineturnover in HeLa cells by lyngbyatoxin A and dihydroteleocidin B. Biochem.Biophys. Res. Commun., 702:100-107,1981.

32. Scott, R. E., Florine, D., Wille, J. J., Jr., and Yun, K. Coupling of growth arrestand differentiation at a distinct state in the G, phase of the cell cycle: Gfl. Proc.Nati. Acad. Sei. U. S. A., 79: 845-849,1982.

33. Shimizu, N., Behzadian, M. A., and Shimizu, Y. Genetics of cell surfacereceptors for bioactive polypeptides: binding of epidemial growth factor isassociated with the presence of human chromosome 7 in human-mouse cellhybrids. Proc. Nati. Acad. Sei. U. S. A., 77: 3600-3604,1980.

34. Shimizu, Y., and Shimizu, N. Genetics of cell-surface receptors for bioactivepolypeptides: a variant of mouse BALBC/3T3 fibroblasts possessing alteredinsulin-binding ability. Somat. Cell Genet., 6: 583-601,1980.

35. Shoyab, M., DeLarco, J. E., and Todaro, G. J. Biologically active phorbolesters specifically alter affinity of epidermal growth factor membrane receptors.Nature (Lond.), 279: 387-391,1979.

36. Shoyab, M., Warren, T. C., and Todaro, G. J. Phorbol-12,13-diester 12-esterhydrolase may prevent tumour promotion by phorbol diesters in skin. Nature(Lond.), 295:152-154, 1982.

37. Slaga, T., Sivak, A., and Boutwell, R. K. (eds.). Carcinogenesis, Vol. 2. New




Tumor Promoters and Adipocyte Differentiation

York: Raven Press, 1979.38. Sugimura, T., Miwa, M., Saito, H., Kanal, Y., Ikejima, J., Terada, M., Yamada,

M., and Utakaji, T. Studies of nuclear ADP-ribosylation. Adv. Enzyme Regul.,18: 195-220, 1980.

39. Takashima, M., and Sakai, H. A. A new toxic substance, teleocidin, producedby Streptomyces. Part 1. Production, isolation and chemical studies. Bull.Agrie. Chem. Soc. Jpn., 24:647-651,1960.

40. Umezawa, K., Weinstein, I. B., Horowitz, A., Fujiki, H., Matsushima, T., andSugimura, T. Similarity of teleocidin B and phorbol ester tumour promoters ineffects on membrane receptors. Nature (Lond.), 290: 411-413,1981.

41. Van Duuren, B. L. Tumor-promoting agents in two-stage carcinogenesis. Prog.Exp. Tumor Res. Mol. Biol., 11: 31-68,1969.

42. Verma, A. K., Froscio, M., and Murray, A. E. Croton oil and benzo(a)pyreneinduced changes in cyclic 3':5'-adenosine monophosphate and cyclic guano-sine 3':5'-monophosphate phosphodiesterase activities in mouse epidermis.

Cancer Res., 36: 81-85,1976.

43. Verma, A., Rice, H., and Boutwell, R. K. Prostaglandins and skin tumorpromotion: inhibition of tumor promoter induced omithine decarboxylase activity in epidermis by inhibitors of prostaglandin synthesis. Biochem. Biophys.Res. Commun., 79:1160-1166,1977.

44. Weinstein, l. B., Yamasaki, H., Wigler, M., Lee, L. S., Fisher, P. B., Jeffrey, A.M., and Grunberger, D. Molecular and cellular events associated with theaction of initiating carcinogens and tumor promoters. In: A. C. Griffin and R.C. Shaw (eds.), Carcinogens: Identification and Mechanisms of Action, p. 399.New York: Raven Press, 1979.

45. Wenner, C. E., Moroney, J., and Porter, C. W. Early membrane effects ofphorbol esters in 3T3 cells. In: T. J. Slaga, A. Sivak, and R. K. Boutwell (eds.),Carcinogenesis, Vol. 2, pp. 363-378. New York: Raven Press, 1978.

46. Williams, I. H., and Polakis, S. E. Differentiation of 3T3-L1 fibroblasts toadipocytes: the effect of indomethacin, prostaglandin E, and cyclic AMP onthe process of differentiation. Biochem. Biophys. Res. Commun., 77: 175-186,1977.

Fig. 1. Morphology of 3T3-L1 cells on Day 6 after various treatments. Phase-contrast micrograph of cells which

were treated for 2 days with Â¡nducers(DEX and MIX) (A),inducers and TPA (100 ng/ml) (B), Â¡nducersand DHTB (10ng/ml) (C), DHTB (10 ng/ml) (D), inducers and insulin (1 ng/ml), (E), inducers, insulin (1 ^g/ml), and TPA (100 ng/ml)(F), inducers, insulin (1 ^g/ml), and DHTB (10 ng/ml) (G), orTPA (1 Â¿.g/ml)(H).

OCTOBER 1983 4979



1983;43:4974-4979. Cancer Res Yoshiko Shimizu, Nobuyoshi Shimizu, Hirota Fujiki, et al. Differentiation of 3T3-L1 CellsPhorbol Ester Tumor Promoters on the Adipocyte Distinct Inhibitory Effects of Dihydroteleocidin B and the

Updated version

http://cancerres.aacrjournals.org/content/43/10/4974

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/43/10/4974To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



distinct inhibitory effects of dihydroteleocidin b and the ... · l1, is capable of differentiating...

Documents