Estrogen induction of progestophilins in rat estrogen-sensitive cells grown in media supplemented with sera from castrated rats and from rats bearing an α-fetoprotein-secreting hepatoma

Experimental

Cell Research

150 (1984) 390-399

Estrogen Induction of Progestophilins in Rat Estrogen-sensitive Cells Grown in Media Supplemented Sera from Castrated Rats and from Rats Bearing an a-Fetoprotein-secreting Hepatoma ANA M. SOTO,‘~* HANMIN LEE,’ JAMES T. MURA12 and CARLOS ‘Department

with

PENTTI K. SIITERL2 SONNENSCHEIN’

of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, MA 02111, and 2Reproductive Endocrinology Center, University of California, San Francisco, CA 94143, USA

The purpose of this work was to study the effect of a-fetoprotein (AFP) over cell multiplication and the induction of an estradiol-178 (E2)-dependent marker, i.e., progestophilins in E-sensitive cells C29RAP derived from a W/Fu rat pituitary tumor. These cells proliferate in isogeneic hosts under the influence of EZ, while they proliferate in culture regardless of the presence of E2. C9RAP cells were grown in medium supplemented with 10% horse serum. Progestophilin levels were measured 48 h after adding serum (20% horse, or castrated rat, or AFP-secreting tumor-bearing rat) and estrogen to the 10% horse serum-supplemented medium in which the cells were growing. Maximal induction of progestophilins was obtained at 3X10- lo M Ez in cells grown in medium containing horse or castrated rat serum. In contrast, maximal induction of progestophilins required 3~ lo-* M E2 in cells grown in medium supplemented with the serum of Morris hepatoma 7777-bearing rats. This serum contained AFP levels comparable to those present at birth in the rat. 1 I-Methoxy-17p ethynylestradiol (R2s5s), a synthetic estrogen with little affinity for AFP, was also tested for its ability to induce progestophilins. The degree of maximal induction of progestophilins expressed as percentage of the respective control, was similar for all experimental groups, both with E2 and with R2s5s. In addition, we compared the free E2 levels in the culture medium with the progestophilin levels and the cell proliferation rate. We found that the progestophilin levels were maximal at free E2 concentrations above 11 pg EJml, whereas there was no correlation between the free E2 levels and the proliferation rate. Moreover, the proliferation rate of cells in medium supplemented with horse or castrated rat serum was maximal at concentrations of free E2 below 0.4 pg/ml, whereas cell proliferation was inhibited with hepatoma serum even at concentrations of free E2 of 44 pg/ml. We conclude that the effect of hepatoma serum on the E2 induction of progestophilins seems to be mediated by the effect of AFP on the availability of free estrogen, since it is abolished by the addition of both natural and synthetic estrogens. The inhibitory effect of hepatoma serum upon cell proliferation is not reversed by estrogens and thus seems to be mediated by mechanisms other than Ez trapping by AFP.

The response to estrogens (E) in mature female rats consists of increased cell proliferation (hyperplasia), increased protein content per cell (hypertrophy) and positive [l] and negative [2] effects upon the synthesis of some specific proteins. In contrast, the estradiol 17/I (E2) response in newborn female rats is incomplete. * To whom offprint requests should be sent. Copyright @ 1984 by Academic Ress, Inc. All rights of reproduction in any form reserved 0014-4827184 $03.00

Estrogens,

progestophilins

and cell proliferation

391

Estrogen administration results in the induction of specific proteins such as creatine kinase [3-51 and progestophilins [6]; oophorectomy results in increased plasma gonadotropin levels [7, 81. On the other hand, the hyperplastic response of the uterus to estrogen administration does not become evident until 15-20 days of age when marked increases of the mitotic indices of all uterine layers are first observed 24 h after a single E injection [3]. Our data obtained using an “in animal-in culture” model indicate that the lack of proliferative response to E in the newborn rat is due to an apparent direct effect of a-fetoprotein (AFP) upon Esensitive cells [9-l 11. This we verified by using pituitary, mammary and endometrial cell lines that develop as tumors in syngeneic hosts, and by observing the features of the intact uterus and vagina of adult rats bearing a hepatoma that secretes AFP in concentrations comparable to those present in rats at birth [II]. Moreover, when AFP was purified from fetal rat serum and tested in culture conditions we observed that the tumor cells CgRAP were specifically inhibited in their ability to multiply, and that this inhibition was not reversed by estrogens

WI. The purpose of this work is to determine how the direct mechanism of induction of specific proteins by estrogens is affected by AFP in a well defined culture system. As a marker we selected the ability of E2 to induce progestophilins [ 131 in the E-sensitive rat pituitary cell line CQRAP [14]. MATERIALS

AND

METHODS

Cells and Media C29RAP is a rat pituitary tumor cell line. These cells are genuine estrogen-sensitive cells: by this we mean that they have estrophilins, they respond to E2 by increasing their progestophilin levels [13], and they are E,-sensitive for cell proliferation when inoculated into syngeneic hosts [14]. FR3T3 is an established Fisher tat tibroblast cell line, used as an E-insensitive control in the cell multiplication experiments. Both cell lines were grown in culture conditions as already described [14] in Dulbecco’s Modified Eagle Medium (DME) (Gibco, Grand Island, N.Y.) supplemented with 10% heat-inactivated (56°C 30 min) horse serum (HS) (Flow Laboratories, Rockville, Md) in T7S Falcon flasks (Coming, N.Y.). BUF rats used for these experiments were purchased from Charles River Breeding Labs (Wilmington, Mass.).

Induction of Progestophilins Progestophilins were measured by in vivo uptake of [3H]promegestone (17,21 dimethyl-19-nor 4,7 pregnandiene-3,2 0-dione) (New England Nuclear Co., Boston, Mass.) after a 48 h induction period [13]. One day after subculture in 10% HS-DME in T75 flasks, groups of ten cultures were treated as follows: addition of heat-inactivated (a) HS to a final concentration of 30%; (b) Morris Hepatoma 7777-bearing BUF rat serum [16] (Hep S) to a final concentration of 20% Hep S- 10% HS; the AFP level in this medium was 3 mg/ml; and (c) addition of heat-inactivated castrated female BUF rat serum (CRS) to a final concentration of 20% CRS - 10% HS. In addition, duplicate flasks in each group were supplemented with: (1) 3 x lo-” M Ez; (2) 3 x IO- 9 M E2; (3) 3x10-s M E2; (4) ~xIO-~ M llmethoxy-17/l ethynylestradiol (Rzsss) and (5) vehicle. The rationale behind using Ez and R 2858 as stimulants of specific protein synthesis in these experiments rested upon the evidence suggesting that while the natural estrogen Ez binds to rat AFP with high affinity [15], the synthetic estrogen Rzs5a does not 1161. R2a5s was obtained through a generous gift by Dr J. P. Raynaud, Roussel-UCLAF, Paris, France. After a 48 h induction period, [‘Hlpromegestone (sp. act. 87 Ci/mmole), at a final concentration of 2.5~10~~ M was added into Exp Cell

Res IS0 (1984)

392 Soto et al. each flask. Controls of unspecific uptake for each experimental group received [3H]promegestone plus-lOO-fold non-radioactive progesterone. The progestin uptake protocol was carried out at 37°C in a 95% air-5% CO2 atmosphere for 60 min. Cells were harvested by shaking, and then they were centrifuged at 800 g for 5 min. Aliquots of the cell-free culture medium were taken for radioactivity determinations. Cells that were attached to the flask were freed with ‘kypsin-EDTA (G&co, Grand Island, NY) for 2 min and then rinsed with ice-cold horse serum supplemented medium to inactivate the trypsin. This cell suspension was added to the respective pellet, and after centrifuging and washing twice with ice-cold phosphate-buffered saline (PBS), the cells were resuspended in 1 ml PBS-2 mM EDTA. The resulting cell suspension was disrupted by sonication (Ultrasonic Inc., Model W185D, Plainview, L.I., N.Y.) at setting 6 for 3 set three times with 30-set cooling periods. Cell disruption was monitored at the light microscope, after staining with toluidine blue. Triplicate aliquots of the homogenate were taken for determination of the radioactivity, DNA content [17] and protein content [18]. To validate the uptake data, some homogenates were centrifuged at 105000 g for 90 min and the [3H]promegestone content was measured both in the cytosol and in the particulate fraction. Aliquots from the cytosol were taken to measure specific binding by charcoal adsorption of the unbound hormone [13, 191. The statistical analysis of the data was performed by analysis of variance (ANOVA) and the Newman-Keul post-hoc test [20].

Comparison of the Multiplication Rate of C29RAP and FR3T3 Cells in Media Supplemented with Different Estrogens and Sera Cells were harvested, resuspended in DME and counted. Aliquots containing approx. 150000 cells were mixed with 10 ml of each of the different medium listed above; 0.5 ml of the cell suspensions thus obtained were seeded per well of Costar 3524 multiplates (Cambridge, Mass.). Cells were allowed to multiply in a 95 % air/5 % CO2 incubator at 100 % humidity and 37°C. Cells were harvested every other day by a short trypsin-EDTA treatment. Cells were collected by centrifugation, and after washing them twice with PBS, they were resuspended in 0.5 ml PBS and stored at -20°C. Cell multiplication rates were estimated by measuring the increase in the DNA content/well as a function of time. Rat sera were obtained from castrated female BUF rats (CRS) and from Morris Hepatomas 7777-bearing castrated female BUF rats (Hep S) fasted for 16 h. Animals were rendered unconscious by short exposure to a CO2 atmosphere, and blood was drawn from the orbital sinus. The pooled blood was allowed to clot a room temperature, and the serum was clarified by centrifugation at 3 000 rpm for 20 min. After heat inactivation at 56°C for 30 min, the serum was filtered through 0.22 pm Millipore filters and was stored at -20°C until needed. AFP levels were determined by rocket immunoelectrophoresis [21] and Et concentration was determined by RIA using the method described by R. Goodman [22] with only minor modifications. Briefly, 0.5 ml serum samples were extracted with ether, chromatographed through Sephadex LH20 with chloroform : heptane : methanol: water (500 : 500 : 75 : 3), and assayed with a ‘sensitized’ method, consisting in the addition of the tracer 30 min before separation of bound and free hormone by charcoal-dextran adsorption. The standard curve ranged from 2 to 20 pg; 50% displacement occurred at 7-8 pg. The water blanks ranged from 1 to 1.5 pg and were subtracted from the values obtained with serum samples of similar volume. The antibody used was Niswender’s antibody no. 244 [22]. Determination of the EZfree/bound ratio was done by the method described by Hammond et al. [23].

RESULTS E2 Induction

of Progestophilins

in C29RAP Cells

A detailed study of the E2 inducibility and the physicochemical properties of progestophilins in CQRAP cells has been published [ 131. A preliminary experiment was done with the purpose of validating the uptake data for [3H]promegestone. Cells were sonified as reported in Methods, and a 105000 g cytosol was obtained. Counting of comparable aliquots before and after charcoal treatment Exp Cell

Res I50 (1984)

Estrogens,

progestophilins

and cell proliferation

393

revealed that less than 20 % of the radioactivity was removed in these conditions. All radioactivity incorporated migrated as a single 8S peak when run in sucrose gradients ([13, 191 and data not shown]. A comparison of the radioactivity present in the homogenate, the 105000 g supernatant, and the one remaining in the particulate pellet revealed that about 70-80% of the total radioactivity was in the particulated fraction. Thus, we chose to express our results as uptake of [3H]promegestone without distinguishing between the cytoplasmic and translocated nuclear progestophilin. It is worth mentioning that although the uptake data was obtained at concentrations of [3H]promegestone below those required to saturate the progestophilins, the correlation between percent induction over control obtained through Scatchard analysis [24] and these uptake experiments is satisfactory [13]. A series of preliminary experiments comparing the dose-response curve for R2s5s and E2 were performed in cultures supplemented with HS and with Hep S. The results showed that the progestophilin maximal levels were achieved with 3x lo-*’ M Ez and 3x lo-” M Rtsss in HS and with 3~ lo-* M E2 and 3x 10-l’ M R2s5s in Hep S, The ratio of the progestophilin levels achieved with 3x 10m8 M E2 over the levels achieved with 3~ 10-l’ M E2 were 1.OOIfIO. 10 in HS and 2.3kO.39 in Hep S. In contrast, the ratio of the progestophilin levels achieved with 3x 10e9 M R2s5s over the levels achieved with 3x lo-” M R2s5s were 1.25f0.04 and 1.07+0.06 in HS and Hep S, respectively. These experiments also showed that both the baseline (unstimulated) levels and the progestophilin levels in maximally stimulated cells were both higher in HS than in Hep S; i.e., the ratio HS/Hep S was 1.98f0.58 for the unstimulated cells and 2.15f0.43 in the cells stimulated with 3x 1O-8 M EZ. The disparity of the baseline and the maximal promegestone-specific uptake/pg DNA between cells in HS and Hep S was tentatively postulated to be species-dependent. If so, these differences may disappear when comparing the promegestone uptake in Hep S with CRS-supplemented cultures. Thus, a series of three experiments designed to compare the specific uptake of promegestone/pg DNA at different E2 concentrations was performed .in cultures treated for 48 h with HS, CRS and Hep S. Data obtained in these experiments are shown in fig. 1. It is apparent that the progestophilin level in maximally stimulated cells, i.e. the ones treated with 3x 1O-8 M E2, is higher in the cells growing in HS than in the cells growing in CRS and in Hep S (~~0.01); this parameter is also higher in CRS than in Hep S (~~0.01). The difference between the progestophilin levels in unstimulated HS and Hep S grown cells were statistically different at the p
ISO(1984)

394 Sot0 et al.

ESTROGEN

CONCENTRATION

(M1

Fig. I. Specific [3H] promegestone uptake of C29RAP cells 48 h after culture in media supplemented with A-A, 30% HS and different E2 concentrations; A, 30% HS+3x 10m9 M Rzsss; n a, 20% CRS- 10% HS and different E2 concentrations; El, 20% CRS-10% HS+3x10m9 M R2s5s; O-O, 20% Hep S- 10% HS and different E2 concentrations; 0, 20% Hep S+3x lO-9 M Rzsss. Fig. 2. Percent induction of progestophilins in Cr9RAP cells 48 h after culture in medium containing A-A, 30 % HS; M-M, 20 % CRS-10% HS; @-O, 20% Hep S-10% HS. Fig. 3. Cell proliferation curves of (A) Ca9RAP cells; (B) FR3T3 cells. O-@, 30% HS; O-D, 20% CRS-10% HS; A-A, 20% Hep S-10% HS; A-A, 20% CRS-10% HS+3xlO-* ME*; CD, 20% Hep S-10% HS+3xlO-s MEa; O-Q 20% Hep S-10% HS+3xlO-’ ME*; and X-X, 20% Hep S-10% HS+3x lO-9 M Rzsss,

treatments among themselves. Comparable results were obtained with cultures grown in CRS. In contrast, the statistical analysis of the progestophihn levels in Hep S-treated cultures showed that: (i) there was no significant difference between the control and 3 x 10-l’ M E2-treated cultures; (ii) there was a significant difference bcO.05) between the progestophilin levels found in cultures treated with 3 x 10e9 M E2 and 3 x IO-’ M E,; (iii) there was no difference between the progestophilin levels found in cultures treated with 3x 10e8 M EZ and 3X 10e9 M R2s5s; (iv) the control was significantly different from the cultures treated with 3~10-~ M EZ, 3x10-* M E2 and 3~10~~ M R2sSs (p
Cell

Res I50 (1984)

Estrogens,

progestophilins

Table 1. Comparison between E2 levels, progestophilin tion rates of C29RAP cells

and cell proliferation induction

395

and prolifera-

E2 Concentration Media 30% 30% 30% 20% 20% 20%

HS HS+3xlO-“‘ME HS+3x 1O-9 ME*’ Hep S+lO% HS Hep S+lO% HS+3x1W9 Hep S+lO% HS+3~10-8

M E2 M E2

% Free J%

Total (pdml)

Free (pi&l)

11.98 12.03 11.53 0.47 0.56 0.44

<4 100 1000 25 1000 10 000

12.0 115.3 0.11 5.6 44.0

% Progestophilin induction

Doubling time (hours)

100 300 300 100 230 320

24 24 24 z-240” >240” >240a

a Doubling time cannot be reliably measured because of the extremely low proliferation fig. 3).

rate (see

Hep S is statistically different from the level achieved with (i) 3x 10m9 M E2 in Hep S (~~0.05); and (ii) with all the hormone treatments in Hep S, CRS and HS (~~0.01). No other significant differences were found among the different groups. In summary, the maximal increase of specific promegestone uptake was achieved in cultures treated with 3x lo-*’ M E2 in HS-supplemented medium. In contrast, maximal induction of progestophilins in media supplemented with Hep S occurs at higher E2 concentrations, i.e., between 3x 10V9 M and 3x lo-* M. Rzgs8-mediated induction of progestophilins is maximal at 3~ 10V9 M, regardless of the presence of AFP in the culture medium. The maximal progestophilin induction achieved by each estrogen expressed as percent increase over respective control was comparable in HS-, CRS- and Hep S-supplemented media. However, when these values were expressed as the absolute amount of specific binding per ug DNA the maximal induction was achieved in cells grown either in R2ss8 or in ET30% HS; here the progestophilin levels were about twice as high as in cells grown in R*ssg-Hep S. Table 1 shows that maximal progestophilin levels are achieved at concentrations of free Ez of 12 pg/ml in cultures growing in HS. The progestophilin levels in cultures grown in Hep S increased as the free E2 concentration increased. The plateau was reached as the free E2 concentration increased from 5.6 to 44.0 r&ml. E2 Effect on Cell Proliferation C29RAP cells proliferate maximally in medium supplemented with as little as 1% pooled adult rat serum [9] as well as in 1% CRS, 3 % HS and 3 % castrated and adrenalectomized calf serum (CACS) (not shown). The maximal E2 concentration in media containing 1% serum did not exceed 0.1 pg/ml, i.e. 3x lo-” M. Since the Kd of the estrophilin-E2 complex is 3x lo-” M in these cells [ 141, it 26-848332

Exp CeNRes

150(1984)

396 Soto et al. seems unlikely that the maximal growth rate observed was due to the action of Et mediated through the cellular estrophilins. Moreover, these cells proliferate at similar rates in serumless medium devoid of E and in Ez-supplemented serumless medium [39]. Fig. 3A shows the multiplication pattern of these cells when challenged to proliferate in media supplemented with or without estrogen and either 30% HS, 20% CRS+ 10 % HS and 20 % Hep S+ 10 % HS. It is apparent that the medium containing AFP inhibits cell multiplication, and that addition of either E2 (3X10-’ M or 3~10~~ M) or R2858 (3x10- 9 M) to the culture medium does not abolish this inhibition. Furthermore, estrogens do not significantly affect the multiplication rate of these cells when added either to cells that multiply maximally, i.e., in 3 % to 30% HS (not shown) or 20% CRS- 10 % HS, or to blocked cells, i.e., those kept in 20% Hep S- 10% HS (fig. 3A). Table 1 also shows that the inhibition of cell proliferation observed in cultures supplemented with Hep S is not reversed by concentrations of free E2 above 44 pglml, whereas cultures supplemented with HS proliferate maximally at concentrations of free E2 below 0.4 pg/ml. E2 EfSect on the Proliferation

of FR3T3 Cells

To further characterize the specificity of the effect of Hep S we used FR3T3 cells, a rat fibroblast cell line which does not carry measurable amounts of estrophilins, and does not induce the formation of tumors at the site of inoculation in syngeneic F344 rats. The results shown in fig. 3B indicate that the multiplication rate of FR3T3 cells is not affected by the media supplemented with any of the above mentioned sera regardless of their AFP concentration and/or their being further supplemented with 3 x 10m9 M Rzs5s and 3~ lo-’ M E2 (fig. 3). Thus, AFP partially inhibits the induction of progestophilins by E2 in C29RAP cells, and this inhibition can be totally abolished by increasing the estrogen concentration. In contrast, AFP blocks the multiplication of these estrogensensitive cells in a specific fashion (does not affect other El-non target cells, i.e., FR3T3 cells); also, this effect is not reversed by addition of either natural or synthetic estrogens.

DISCUSSION Induction of the synthesis of specific proteins by estrogens seems to be affected directly by these hormones, since this effect can be demonstrated both in animals as well as in culture conditions [2, 9-l 1, 251. The proliferation of normal and tumor estrogen-sensitive cells in vertebrates is, however, likely to be controlled through an indirect pathway, since E2 does not affect per se the multiplication rate of these same E-sensitive cells in culture [13, 26, 271. In fact, Esensitive cells multiply at their maximal attainable rate in serum-supplemented media even in the presence of undetectable E2 in the culture medium (lo-r3 M). Exp Cell

Res 150 (1984)

Estrogens,

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Moreover, Barnes & Sato [28], and more recently Butler et al. [29] have shown that when genuine estrogen-sensitive cells are grown in a serumless medium devoid of any estrogenic hormones, their growth rate is not affected by the addition of estrogens. The above-mentioned data constitutes the basis for our hypothesis postulating that the initiation of cell multiplication is under negative control, i.e., the ability of these cells to multiply would be a dominant character that could only be regulated through repression. We have obtained data compatible with this working hypothesis in this system [9, 12, 26, 271, in other rat Ezsensitive tumors [lo, 111, in quail oviduct [30] and in rat fibroblasts [31]. The purpose of this work was to determine whether AFP affects both the direct (protein induction) and indirect (proliferation) estrogen effects. We chose to study the effect of E2 over the cellular concentration of specific promegestonebinding sites. Although factors other than an increase of the rate of progestophilin synthesis could affect the specific promegestone uptake, compelling circumstantial evidence of the binding data supports this interpretation [32, 331. We used unfractionated Hep S as a source of AFP, since 23% of the total protein content is AFP, and because of the experimental evidence showing that E-sensitive cells display similar behavior when inoculated into newborn animals [9, lo] and Morris Hepatoma 7777-bearing rats [l I], and in cultures supplemented with purified AFP [ 121. The promegestone-specific binding data shows (fig. 1) that the E2 dose-response curves reach higher values in HS than in CRS, and in turn, it is higher in CRS than Hep S. These data indicate that both CRS and Hep S may contain factors that negatively affect the specific binding of promegestone, or conversely, that horse serum contains factors that affect positively this parameter. Whether this effect is due to interference with the progestogen-binding site, or rather it operates through the synthesis of progestophilins cannot be ascertained from the data currently available. Hep S, however, seems to interfere with the Ez effect upon progestophilin levels by shifting the maximal E2 response two orders of magnitude, i.e., from 3x10- lo M, as shown to occur in cells grown in HS and CRS, to 3~ lOMa M. A possible explanation for these results is that AFP binds Ez with high affinity, thus decreasing the level of free hormone which according with the current prevailing opinion might be the active form of the hormone. Further support for this inference is indicated by a direct relationship between the level of free E2 and the degree of induction of progestophilins in cells grown in Hep S, as shown in table 1. This seems to be confirmed when R2858, a synthetic E that binds poorly to AFP, was used as inducer; the concentration at which full induction of progestophilins was attained, was similar for both AFP supplemented and control HS supplemented media. AFP blocks the multiplication of E-sensitive cells both in the animal and in culture (fig. 3A) [9-121. E-insensitive cells are not affected (fig. 3B). This result strongly suggests that the effect is specific to E-sensitive cells, rather than a nonspecific toxic effect. Although we cannot rule out the presence of inhibitors of the Exp Cell

Res 150 (1984)

398 Sot0 et al. multiplication of E-sensitive cells other than AFP in Hep S, we attribute this negative effect upon cell proliferation to AFP, based on our published evidence, whereby purified AFP specifically inhibited the proliferation of C29RAP cells in culture [12]. This inhibitory effect seems not to be mediated through ‘trapping’ of estrogens by murine AFP as it has been previously suggested [34], since it cannot be reversed by the addition of E2 in amounts that increase the concentration of free E2 above the physiological levels, i.e., 3&300 pg/ml of free hormone. Moreover, R2s5s, which maximally induces progestophilins at similar concentrations, regardless of the presence of AFP, failed to reverse the cell multiplication inhibitory effect of AFP. In addition, maximal proliferation rates of C29RAP cells were observed both in media supplemented with 10% HS-20% CRS at concentrations of free E2 below 1 pg/ml (fig. 3) and in medium supplemented with 3% castrated and adrenalectomized calf serum, which total E2 concentration approaches lo-l2 M. Although the presence of estrogens other than E2 in the sera used to supplement the culture media cannot be ruled out, it seems unlikely that the maximal proliferation rate observed both in HS and CRS would be caused by these estrogens, because E2 does not increase the proliferation rate of E2sensitive cells when added to chemically defined serumless medium [28, 291. Thus, while the effect of AFP upon protein synthesis is reversed by estrogens and seems to be due to the estrogen-binding properties of murine AFP, the effect of AFP upon cell multiplication is not abolished by estrogens, pointing once more to the independence of the two phenomena. These results are comparable to those obtained in newborn animals, whereby induction of specific proteins by estrogens is operative since early postnatal life in rats [3-121, whereas E-induced cell multiplication is not affected by E2 while AFP levels are high [3, 5, 9-12, 351. This seems to be so not only in murine species where AFP binds estrogens with high affinity, but it also holds true in guinea pigs and hamsters, species in which AFP does not bind to estrogens [36, 371. It is worth noting that AFP in the native state is bound to ligands other than estrogens, such as unsaturated fatty acids [38]. At the present it is not known whether the inhibitory effect of AFP upon cell proliferation is due to the protein moiety itself, the AFP ligands, or both. In summary, the data presented herein suggest that: (1) Cell multiplication and synthesis of specific proteins need not be associated; in fact, the control of these two mechanisms is likely to be exerted through independent pathways. (2) Cell multiplication seems to be a constitutive character of these cells which expression is amenable to control through repression. (3) AFP and/or its ligands appears to be a physiological inhibitor for the multiplication of E-sensitive cells during perinatal life. We are presently working on clarifying the molecular aspects of this phenomenon. The authors wish to recognize the technical aid provided by John T. Papendorp, the helpful discussions with Dr D. Damassa and the typing and editorial help of MS Cindy Welch and MS Susan Cahoon. This work was supported by USPHS Grant NIH CA 13410. Exp CeNRes

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Received April 22, 1983 Revised version received September 12, 1983

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