morphological effects and metabolism of the molting hormone in aedes aegypti cultured cells

Insect Biochem., 1976, Vol. 6, pp. 433 to 439. Peroamon Press. Printed in Great Britain.

MORPHOLOGICAL EFFECTS AND METABOLISM OF THE MOLTING HORMONE IN AEDES AEGYPTI

CULTURED CELLS

EPHRAIM COHEN, NAOMI LANIR, and ELA ENGLANDER Department of Zoology, The George S. Wise Center for Life Sciences,

Tel Aviv University, Tel Aviv, Israel

(Received 13 February 1976)

Al~Iraet--Growth of Aedes ae#ypti cultured cells was arrested by ct- and ~-ecdysone at concentrations of 0.01 to 10.0 #g/ml. The inhibitory effect was accompanied by increased cell volume. Prolonged exposure of at least 24 hr to the molting hormones was necessary to induce the above effects. 3H- ct-ecdysone was incorporated into the mosquito cultured cells, and 2.1% of the total label added could be detected in thoroughly washed cells. Thin-layer chromatography of the cellular butanolic extract revealed one apolar peak only and no traces of the original labeled ~t-ecdysone or its immediate hydroxylation product, e.g. fl-ecdysone. It is suggested that the hormone was rapidly converted to a metabolite which did not correspond with the apolar 3-~-dehydro-ecdysone, as was initially speculated.

INTRODUCTION

INSECT tissue cultures have been employed in research on effects and mode of action of insect hormones (MARKS, 1970). In vitro studies on hormonal action concerning development and differentiation were car- ried out on organ cultures such as imaginal disks (OaERLANDER, 1969; FRISTROM, 1972), gonads (YAGI et al., 1967; KAr~mVSELLm and WILLIAMS, 1971) and epi- dermis (Agui et al., 1969; MARKS and LEOPOLD, 1970). Other investigations involve effects of hormones on the migratory activities of blood cells (JUDY and MARKS, 1971) and cells from tissue explants (MARKS et al., 1967; JUDY, 1969). Dipteran salivary gland cells are very useful in research on the mode of action of hormones at the chromosomal level (ASHBURNER, 1970).

Insect cell lines, in particular those which can be maintained and subcultured on vertebrate serum con- raining media, can offer a more simple and defined system to be employed in the study of insect hormones. Established insect culture cells have under- gone profound alterations with regard to enzymatic systems, cell membranes and polyploidy (BROOKS and KtmTrl, 1971). Yet they maintain some fundamental cellular features of insects such as thermolability of the ribosomal large subunit RNA and the inability to form the sterol ring system (CottoN and GILBERT, 1975).

In the process of adaptation to grow and multiply in artificial media, insect cultured cells, like many mammalian cell lines, became growth-hormone inde- pendent (GP, ACE, 1958). Nevertheless, several insect cell lines were examined as to their response towards insect hormones. The effect of the molting hormone

seems to be dose-dependent, increasing cellular multiplication rate (MITSUnASH[ and GRACE, 1970; COUR- GEOr~, 1972), cell volume and nucleoside uptake and breakdown (REINECKE and ROBBINS, 1971). When ele- vated quantities were used, the effect on cellular growth was inhibitory (MrrsunAsm and GRACE, 1970; COURGEON, 1972). Also, high doses of molting hormone inhibited cell movement and mitoses in cockroach leg embryonic cells (MARKS et al., 1967) and differentiation of wing disks (DurKOWSKY and OBER- LANDER, 1974).

No information is available so far as to the metabolism of molting hormone in insect cell lines. ~-ecdysone is rapidly hydroxylated to fl-ecdysone in various insects (MoRIYAMA et al., 1970; Cm~BAS and C-'ma~AS, 1970; KING, 1972; HOFF~.NN et al., 1974). Both hormones are metabolized and inactivated by conversion to 3-dehydro analogs or 3-epimers (KhRL- SON and KOOLMAN, 1973; KARLSON et al., 1972; Svo- BODA et al., 1975), by sulphate and glucoronic acid conjugates (HEINRICH and HOFFMEISTER, 1970; KOOL- MAN et al., 1973) or by cleavage of the steroids side chain (MORIYAMA et al., 1970).

The present communication deals with the effects of the molting hormone on growth and morphology in a cell line established from Aedes aegypti embryonic tissues (PELEG, 1969). In addition, the fate of tritiated ~-ecdysone taken up by the cultured cells was investigated.

MATERIALS AND METHODS

Cultured cells

A cell line established from embryonic tissues of Aedes aegypti by PELEG (1969) was grown at 28°C. Stock cultures

t... 6/4+F 433

434 EPHRAIM COHEN, NAOMI LAN1R, AND ELA ENGLANDER

were maintained in disposable plastic culture flasks and subcultured every 7 days. For experimental purposes cells were transferred to plastic petri dishes and kept under humidified 5% CO2 atmosphere. The incubation medium contained fetal calf serum, but no insect haemolymph (PELEG, 1969).

Hormones

Morphological effects. Insect molting hormones (~-ecdysone and fl-ecdysone) were dissolved in the salts solution of the culture medium, sterilized by passing through a Mil- lipore filter and kept in the refrigerator at 4°C until used. Cultured cells were exposed to various quantities of the hormones and effects on morphology and rate of growth were observed daily.

Cell growth. Cell cultures in petri dishes (3.6 cm in diameter) were grown for various periods of time in the presence of fl-ecdysone at a concentration of I pg/ml. When control plates became confluent, the incubation medium was removed from all cultures and the cells were washed three times in a physiological solution (0.11 M NaCI; 0.007 M KC1; 0.001 M NaHCO3; 0.0007 M KH2PO4; 0.0009 M CaC12; 0.03 M sucrose). The washed cells were subse- quently lysed in 1.0 ml of Tris-SDS (0.005 M Tris; 0.1 M NaC1; 0.5% SDS) and kept overnight at room tempera- ture. The lysate was thoroughly mixed on a Vortex, aliquots were removed and protein content was measured using Lowry's procedure.

Metabolism Cells cultured in disposable petri dishes (6.0 cm in dia-

meter) were exposed to a high specific activity 3H-ct-ecdysone (2.0 Ci/m-mole). At the end of the incubation period the medium was decanted and saved. The cells were washed five times in the saline solution described before and collected by using a 'policeman'. The washed cells were suspended in 1.0 ml of the saline and homogenized in a whole glass hand homogenizer. Aliquots of medium as well as cell homogenate were removed, solubilized in Soluene TM-100 (Packard) and directly radioassayed in a Packard Scintillation Spectrometer (Model 3375) using a toluene based scintillation liquid. Cell homogenate and growth medium were separately extracted in butanol and the extracts subjected to Silica gel-G chromatoplates contain- ing a fluorescent indicator (DC-Karten, SiF Riedel-de H~ien). The plates were co-chromatographed with cold

7-ecdysone, using chloroform-methanol (85:15) as a deve- loping system. Location of ct-ecdysone was observed under u.v. light and plates were radioassayed in a Packard Radiochromatogram Scanner (Model 7200).

As reference for ct-ecdysone, fl-ecdysone and their respec- tive 3-dehydro analogs, forth instar larvae of Locusta migratoria were injected with radioactive 7-ecdysone and the steroids were extracted, following essentially the procedure of HOFFMANN and KOOLMAN (1974).

RESULTS

Phase-contrast microscopy of normal and molting hormone-treated mosquito cultured cells (Fig. 1) reveals that exposure to ecdysone resulted in arrested cell multiplication and marked morphological changes, such as enlarged cellular volume and preven- tion of the formation of the characteristic monolayer.

In ecdysone-treated cultures the feeder layer and the small spherical cells that appear upon confluency (PELEG and SHAHAR, 1972) were not seen. The difference in cellular growth could be demonstrated with both ct- and fl-ecdysone at concentrations ranging from 0.01 to 10.0 ~g/ml. Lower doses did not appar- ently affect cellular growth and morphology.

To observe the above hormonal effect the growing cells must be exposed to eedysone for at least 24 hr (Table 1). Cultures were treated with/%ecdysone (l.0 pg/ml) from the time of inoculation until 24 hr before the controls reached confluency. The amount of protein was determined, and served as a measure of cellular growth. It became evident that when cultures exposed to fl-ecdysone for only 24 hr were examined, total protein resembled the control value e.g. 522 /~g protein per plate in treated cultures versus 501 pg proteins in the control. It was realized that prolonged cultivation in the presence of the molting hormone was needed to bring about growth retardation. It is noteworthy that reduced protein quantity was basi- cally the same for cultures exposed to the hormone for 2 to 4 days, and was about 55% of the control.

Table 1. Effect of fl-ecdysone on cellular growth of Aedes aegypti cultures

Addition of hormone Protein per plate Treatment (day) #g _+ S.E. % of control

Control - - 501 _ 4 100 fl-ecdysone* 0 264 + 36 52.7 fl-ecdysone 1 294 ___ 16 58.7 fl-ecdysone 2 277 + l l 55.3 fl-ecdysone 3 522 +_ 36 104.2

Aedes aegypti cells were grown in disposable petri dishes (3.6 cm in diameter) and fl-ecdysone (1 /~g/ml) was added to the incubation medium at different periods following the onset of the cultures. On the fourth day when control plates reached confluency the medium was decanted and the cells were washed three times in a saline solution. The washed cells were lysed in Tris-SDS buffer (0.005 M Tris; 0.1M NaC1; 0.5% SDS) aliquots were removed and the amount of proteins were determined by the Lowry method.

* Addition of fl-ecdysone at the time of inoculation.

.435

Fig. 1. Aedes aegypti cultured cells 5 days after inoculation ( × 300). (A) Control cells. (B) Cells treated with 1/~g/ml/3-ecdysone 2 days after inoculation. Note the enlarged cell volume.

Effects of molting hormone on Aedes aegypti cell line 437

This suggests that-the mosquito cells were more sensitive at a certain period of growth, presumably 1 to 2 days before the formation of a monolayer.

To measure the uptake of 3H-u-ecdysone by Aedes aegypti cultured cells the labeled hormone was added on the third day following inoculation. On the seventh day the cells were harvested and thoroughly washed in saline solution. Aliquots of the washed cells and incubation medium were radioassayed. The medium and the cells contained 1.1 x 106 counts/min and 2.3 x 104 counts/rain respectively. 2.1% of the total radiolabel added to the medium was taken up by the cultured cells. The butanolic extract of the thoroughly-washed cells contained only one major apolar metabolite with a R f value of 0.65 (Fig. 2). The medium extract contained the radioactive ct-ecdysone only, (Rf = 0.23), which remained unchanged. It is plausible that ct-ecdysone entering the cells was rapidly converted to the metabolite so that even traces of the original label could not be detected. fl-ecdysone, which has been shown to be the immediate hydroxylation product of g-ecdysonc in various

2=Ecdysone Medium

. 0 ¸

" 0

n -

C m .----4~

Fig. 2. Thin-layer radiochromatogram of the butanolic extracts of medium and mosquito cultured cells treated with tritiated =-ecdysone. Three-day old cultures were exposed to tritiated ,,-ecdysone for an additional three days. Both medium and thoroughly washed cells were extracted in butanol, and aliquots were applied to silica gel-G chromatoplates. The plates were developed with Chloroform-methanol (85 : 15) and radioscanned from left to right. The R r values for u-eedysone and for the metabo-

lite detected in the cells are 0.23 and 0.65 respectively.

in vivo and in vitro systems (KARLSON et al., 1972; Hv.tNRICH and HOFFMEISTER, 1970), could not be found either.

It was initially thought that the metabolite might be an oxidation product of ~-ecdysone. In order to elucidate this point the migratory locust forth instar larvae were injected with 3H-~t-eedysone (10/zCi/ani- real). Four hours later, total body steroids were extracted and examined according to HOFFMANN and KOOLMAN (1974). The major four peaks of ~t-ecdysone, fl-ecdysone, and the corresponding nonpolar 3-dehydro derivatives were detected. When our metabolite was co-chromatographed with the locust's ecdysones, its migration distance was shorter than the 3-dehydro-~-ecdysone and turned out to be more polar in comparison to the latter. It is not clear whether ~-ecdysone or its metabolite induced the observed cellular response.

DISCUSSION

Both stimulatory and inhibitory effects of molting hormone have been reported for insect tissues and cultured cells. It appears that these effects are to some degree dose dependent. Low concentrations of ecdysone were found to increase cell multiplication rate in Antheraea eucalypti (MITSUHASm and GRACE, 1970) and Drosophila melanogaster (COURGEON, 1972) cell lines, while higher doses appear to suppress cell growth and development in the same cultured cells. COUR~EOIq (1972) describes a differential effect exerted by or-and fl-ecdysone. The former was inhibitory at a relatively high level in comparison to the latter. This discrepancy was probably related to the poor solubility of ct-ecdysone in aqueous solutions and to the reduced penetration through cell membranes. COHEN and GILBERT (1972) observed that two lepidopteran cell lines did not take up ~t-eedysone whereas fl-ecdysone penetrated to a limited extent. In contrast to the above cell lines, mosquito cultured cells were capable of absorbing considerable amounts of ~-ecdysone (2.1% of the label added to the medium). This uptake might account for the arrest of cell multiplication and the morphological changes. We could not find any stimulatory effects of ~- and fl-ecdysone, using low doses and levels above 0.01 #g/ml were always inhibitory.

It is widely accepted that the molting hormone is implicated in stimulation of imaginal disks development (OBERLANDER, 1969; FRISTROM, 1972), cuticle deposition (AGuI et al., 1969), differentiation of male gametes (YAGI et al., 1969; KAMBYSELLIS and WIL- LIAMS, 1971), increased migratory activities of blood and tissue explant cells (MARKS et al., 1967., JUDY, 1969) as well as stimulation of RNA and protein synthesis (CONGOTE et al., 1969; WYATT, 1972). Neverthe- less, reports are being accumulated which point also to some inhibitory effects of the steroidal hormones.

438 EPHRAIM COHEN, NAOMI LANIR, AND ELA ENGLANDER

Dose dependent effects were realized in organ cultures such as in cockroach embryonic leg (MARKS et al., 1967) where doses of fl-ecdysone in the range of 0.1 to 0.5 /lg/ml were stimulatory whereas higher levels arrested cell movement and mitoses. Recently similar doses of hormone were reported to affect cuticle deposition in wing disks of a lepidopteran species (DUTKOWSKY and OBERLANDER, 1974).

fl-Ecdysone could initially decrease RNA synthesis in third instar Calliphora larvae (NEUFELD et al., 1968) and inhibited to a large extent RNA formation in germinal cysts of Hyalophora pupae (COHEN and GIL- BERT, 1974). The interpretation of the above-men- tioned controversy is a matter of speculation. Sup- pressed RNA synthesis is not necessarily in conflict with an overall stimulatory action of the molting hormone. It may be essential and related to some control mechanism whereby certain RNAs are degraded and other RNAs are induced and protected, as postulated by COHEN and GILBERT (1974). The physiological sig- nificance of growth arrest of cultured cells and organs by treatment with fl-ecdysone is not presently under- stood. It can be speculated that since inhibitory effects are in most cases displayed at high levels of the hormone, that an excess of the inducing agent might in- terfere with normal balance of developmental pro- cesses, and the cell system is unable to inactivate and eliminate the steroids at an appropriate rate.

To induce morphological changes and growth arrest, the mosquito cultured cells have to be exposed for at least 24 hr to ecdysone (Table 1). Short term treatments also failed to exhibit any effects in Anther- aea eucalypti and Trichoplusia ni cell lines with regard to macromolecular synthesis (COHEN and GILBERT, 1972). Short period exposure to ecdysone is appar- ently insufficient to affect cell division and hence no difference between treatment and control could be realized. Prolonged exposure may result in the increased probability that the cells will be affected at the sensitive phase of the cell cycle. The phenomenon of increased cell volume due to ecdysone can be attri- buted to modifications in cell membrance permeabi- lity and also to the capability of non-dividing cells to proceed and produce cellular components.

Thin layer chromatography of the butanolic extract of the cells reveals only one major metabolite. It is noteworthy that no ~-ecdysone could be detected, pointing to a rapid metabolism of the hormone upon penetrating the cells. In other systems where metabolism of ct-ecdysone was followed, it has been demonstrated that several metabolites both polar and apolar, could be observed. Hydroxylation to fl-ecdysone was widely detected as an immediate metabolism product of c~-ecdysone in insects such as Calliphora (KARLSON and KOOLMAN, 1973), Locusta (HOFFMANN et al., 1974), Antheraea (CHERBAS and CHERBAS, 1970) and Hyalophora (GORELL et al., 1972), and in isolated abdomens of Bombyx (MoRIYAMA et al., 1970). Both insect ecdysones were reported to be inactivated by oxidation to the 3-dehydro analogs (HOFFMANN et al.,

1974; KARLSON and KOOLMAN, 1973) or conversion to the corresponding epimers (SvOBODA et al., 1975). Since our metabolite in nonpolar it was suggested to be 3-a-dehydroecdysone. Yet when the compound was co-chromatographed with the methanolic extract of Locusta, it migrated somewhat slower than the 3-~- dehydroecdysone produced by the locusts, ruling out this possibility. The apotar metabolite might be an inactivation product of the mosquito cultured cells. However, the possibility cannot be excluded that this material rather than ct-ecdysone is responsible for the inhibitory effects.

Acknowledgements--I wish to express my thanks to Dr. J. PELEG of the Israel Institute for Biological Research, Ness-Ziona for a starting culture of Aedes aegypti cells. Professor P. KARLSON, Phillipps Universit/it, Marburg, Germany, is gratefully acknowledged for the gift of the tritiated cz-ecdysone.

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Key Word Index: Aedes aegypti cell line; ecdysone; ecdysone metabolism

morphological effects and metabolism of the molting hormone in aedes aegypti cultured cells

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