Among the various p~lyp~~tide and steroid hormones that influence mammary cell prol~eration, cytodifferentiated function, and ne~pl~stic f~nsfo~atiou, the ovarian steroid estradio~ fE2) has been noted to exert a profound in~uen~e on normal as well as t~nsfo~ation-associated prol~eration of the rn~rn~y epithefiaf eell,‘-3 The ovarian steroids, E, and progesterone, in concert with the poly~pt~d~ hormones, ins~i~ and prolactin, and the gluc~o~~~oids induce casein gene expression and secretory activity.3d Under the influence of estrogens, initiated cells produce atypical hyperplasias that are at high risk for deve~opi~ rn~rn~~ caneert7n8and mamm~y tumors regress on ablation of ovarian function .9*%* Address reprint requests to Dr. Nitin T. Telang at the S~~~o~eil ~~~~~~~~ch Laboratory, 510 E. 73rd Street, New York, NY Received S&ember 2, 1991; accepted December 2, 1991.
In vivo studies of spontaneous and/or ~~~inogeninduced rodent mammary can~e~,3~d~~~9-11 however, provide little ~ppo~unity for a mechanistic approach to analyze direct response of the target cells to initiators and rn~~~tors of neoplastic trausformation~ As an alternative approach, in vitro models developed from rn~rn~ epithe~al tissue have been used to identify molecular, metabolic, and cellular markers that are pe~nrbed due to initiation, and are susceptible to modutation by prototypic ~hemoprev~~tive ~ents,1z-~8 Recently we demonstrated that mammary epitheli~ cells from the low cancer risk BALBlc mouse can be tumorigenically t~~sfo~ed when transfected with ras and myc o~~ogenes.~~,~?Prior to the expression of fully transformed tumor cefl phenotypes the oncogenetransfected cells exhibit persistent expression of ras and myc t~~sc~pts, upre~ation of E, biotransfo~a~ tion via the C-160! hydroxylatio~ pathway, increased frequency of ancho~ge-independent growth, and a high incidence of hy~~lasti~ ~~t~owths from transplants. 16~17 Since all these end points precede the emer-
Estrogen de~e~de~~e of mouse mammae Table 1
Morphologic characteristics of mammary epithelial cells Origin
Cell line MMEC
tumor celfs: Suio et al,
MorphologV
Mammary tissue from virgin, female BALE/c mouse
Epithelioid colonies, polygonal cells. Form ‘normal’
ducts in perenchyma-free mammary fat pads. pHO6-f
MMEC transfected with the ras oncogene
T,/Pr,
Primary tumor from pH66T
In vitro morphology similer to MMEC. Form hyperplastic ducts in m8mm8~ fat pads. In vitro morphology similar to MMEC. Form rapidly growing tumors in mammary fat pads and at subcutaneous sites.
@Summarized from refs. 16 8nd 17.
gence of tumors, they can be considered as intermediate biomarkers for preneoplastic t~sfo~ation. In the present study, mammary epithelial cells derived from the BALBic mouse strain are used to induce tumorigenic transformation by the rus oncogene. The rus-transfected, tumorigenic cell is further characterized in vitro for the persistence of endocrine responsiveness and of tumorigenic phenotype. Furthermore, these perturbed biomarkers are used as specific end points to examine the modulatory infhrence of indole3-carbmol WC), a naturally occurring, potentially chemopreventive agent. Experimental
Ceil lines and chemicals The parental mouse mammary epithelial celi line (MMEC) and the rar oncogene transfectants were developed in the Breast Cancer Research Laboratory.i6 The characteristics of the cell lines MMEC (parental), pHO6T (rus transfected), and T,Pr, (tumor cell line derived from pHO6T) are provided in Table 1. Primary tumors from transplanted pHO6T cells were surgically excised, finely minced, and digested by incubation in a 1: 1 v/v mixture of collagenase-hyaluronidase according to published procedures. 15-17The cultures were plated in RJO-mmtissue culture plates and maintained in a humidified atmosphere of 95% air : 5% CO2 at 37 C using the DMEIF-12 medium (see below). The epithelioid colonies that grew were trypsinized and expanded in 25-cm2 flasks using the medium supplemented with 10% fetal bovine serum (FBS), These were designated as T,/Pr, cell line. The T,/Pr, cells at passage 80-90 were used for the experiments. Routinely, all the cell lines were maintained in Dulbecco’s Modified Eagle’s Medium F-12 (DMEJF-12, Sigma Chemical Co., St. Louis, MO, USA) supplemented with 10% FBS (heat inactivated), 4 mM L-glutamine, 100 U/ml penicillin, 100 pglml s~eptomycin, 100 ~glml gentamicin, and 5 pg/ml insulin (Eli Lilly Inc., Indianapolis, IN, USA), with phenol red as pH indicator. Estradiol, tamoxifen (TAM), and 13Cwere obtained from Sigma Chemical Co., and 13Hlthymidine (specitic activity, 40 to 60 Ci/mmol) was obtained from DuPont-NEN, Beverly, MA, USA. The concentrations of stock solutions of E2 and TAM were 1 x 1W* M, and of 13C were 5 x 10-l M.
by sequential reduction of FBS levels to 7.5%, 5.0%, 2.5%, and 1.0%. The cells that survived this selection procedure were evidently capable of growth in reduced levels of serumassociated endocrine factors, including estrogens. For the experiments on estrogen responsiveness, the stock culture was maintained in medium supplemented with 1% serum and 8.63 mg/L phenol red. Cell growth and proliferative activity were determined in serum-free condition with or without exogenous phenol red and Er .
~rawth kineti&s The cells were seeded at an initial density of 1 x 10s cells into T-25 flasks. After an attachment period of 18to 20 hours, the cultures were exposed to the test compounds for 2 to 5 days, depending on the specific experiment. At the end of this exposure, cell number and viability were determined by hemocytometer counts after staining the cells with trypan blue. The data were generated from at least six repiicate experiments.
~~H]~~ymidi~e uptake To determine the extent of rH]thymidine incorporation, the cells seeded at a density of 1.0 X 10scells were treated with noncytotoxic doses of estradiol and were exposed for the last 24 hours of culture to rH]thymidine (5 c~Ci/ml).The cultures were washed twice with cold phosphate-buffered saline to remove the unincorporated thymidine and lysed by exposure to 1 ml 1 N NaOH and 1% sodium dodecyl sulfate made up in phosp~te-bread saline. A total of 500 ~1 of the cellular lysate was then used to determine the ceUular uptake of 13Hlthymidine by liquid scintillation.13J4
Anchorage-independent
growth
To each well of a six-well plate was added 2 ml of the medium containing 0.6% agar (D&o, Nobel Co., Lincoln, NE, USA) to prepare the basal layer of agar. After an 18- to 20-hour incubation for soli~~tion of this layer, 2 ml of the medium containing 0.33% agar, 10% FBS, and 1 x 10’ dispersed cells was overlaid in each well. The plates were incubated for 14 days at 37 C in a humidified atmosphere of 5% CO* : 95% air, and the number of colonies ~40 pm formed was determined. ‘w’
Estrogen metabolism Adaptation of T&Pr, cells for growth in serumfree condition The effect of estrogenic agents was examined on the cells that were initiallyadapted to grow in serum-depletedmedium
The extent of e&radio1 rne~~~srn in the cells via C-17 oxidation, C-2 hydroxylation, and C-16~ hydroxylation pathways was determined by a radiometric assay. 1r*16$r This assay measures 3H exchange from stereospecifically labeled Er to form
Steroids, 1992, vol. 57, June
262
Papers
Table 2
Induction of hyperproliferation
and acquisition
of anchorage-independent
growth by ras oncogene
in mammary
epitheiial
cells Cell line
Population doubling timea
MMEC (parental) pHOST (ras transfected) T,/Pr, (ras transformed]
Number
27.8 rt 2.3c 22.0 it 1.8e 16.7 t 1.79
of anchorage-independent
coloniesb
3.7 zk 1.2d 150 t 28’ 335 2 46h
a Calculated from the linear portions of the growth curves. Mean 2 SD, n = 6. b Tridimensional colonies at day 14 per lo4 ceils plated in 0.33% agar. Mean rt SD, n = 18. c-e P = 0.004; c-9P = 0.001; d-fP < 0.0001; and *h P < 0.00001.
3H20. The amount of )HzO formed provides an indirect assessment of conversion of E2 to estrone (Et), and subsequently to Z-hydroxyestrone (2-OHE,) or to lfk-hydroxyestrone (ItSOHEl) in a stoichiometric manner.t9 The cells were incubated with [ 17-‘H]Er , [2-3HlE,, or [16&H]E2 (5 x lo6 dpm; specific activity, approximately 20 Ci/mmol) for 48 hours. Aliquots (500 ~1) of the medium were diluted to 3.5 ml with water and lyophilized, and sublimed water was counted in a liquid scintillation counter for ‘I&O formation. The extent of metabolism was corrected for nonspecific 3H exchange obtained from the incubations without the cells.
Statistical
analysis
The data were analyzed for their statistical significance by the two-sample t test where appropriate. Probability values of co.05 were considered significant.
Results Growth characteristics The initial experiments were designed to determine growth characteristics of the parental cell line MMEC,
the rus oncogene-transfected pHO6T, and the tumorigenie phenotype TJPr, by examining the alterations in population doubling times and the ability to undergo ancho~ge-inde~ndent growth. Population doubling times were found to decrease by approximately 21% and 40% in pHO6T and T,/Pr, cells, respectively, relative to that seen in MMEC. This decrease signifies enhancement of cell proliferative activity in the mammary epithelial cells that are expressing the transfected rus oncogene. Corresponding to the increased cell proliferative activity, pHO6T and T,/Pr, cells also exhibited a substantial increase in ~cho~ge-independent growth as evidenced by greater number of tridimensional colonies formed when suspended in semisolid medium containing 0.33% agar (Table 2). Estradiol responsiveness Persistence of the response of mammary epithelial cells to E, was evaluated by measuring the relative extent of E, metabolism via the C-17 oxidation, C-2 hydroxylation, and C-l& hydroxylation pathways. In general, estradiol metabolism was elevated in pHO6T and T,/ 264
Steroids,
1992, vol.
57, June
Pr, cells. The data presented in Figure 1 demonstrate that relative extent of C-17 oxidation of estradiol was elevated approximately two-fold in pHO6T and threefold in T,/Pr, cells relative to that in MMEC. The C-2 hydroxylation pathway was found to be downregulated in the pH06T cells but not in the T,/Pr, cells. It was interesting to note that the relative extent of estradiol metabolism via the C-I6a hydroxylation pathway exhibited a three-fold increase in ras-transfected cells and a 43-fold increase in rus-t~nsfo~ed cells. To further confirm the estrogen responsiveness, stock T,/Pr, cells were adapted to grow in serum-depleted (1% serum) medium containing phenol red. These cells routinely exhibited approximately 14-to 16fold increases in cell number within 5 days of culture. In contrast, cells grown in 1% serum without the estrogenie agent, phenol red, exhibited only a four- to fivefold increase in cell number. Thus, deletion of phenol red brought about a 73% decrease in growth of T,/ Pr, cells. A similar response to phenol red was also detected in cultures maintained in serum-free condition. As expected, in serum-free and phenol red-free condition T,/Pr, cells exhibited a nine-fold decrease in cell number at day 5 postseeding relative to the control (1% serum, phenol red-free medium). The addition of phenol red resulted in a 1Cfold increase in celI number. Similar to phenol red, E2 at a dose conventiona~y used in cell culture studies was also able to reverse the growth suppression caused by the deletion of serum and phenol red, as evidenced by an increase in cell number as well as in [3Hlthymidine uptake (Figure 2a,b). Thus, induction of growth by the estrogenic agent phenol red and by the natural estrogen, E2, provides strong evidence that T,/Pr, cells retain estrogen responsiveness. Effect of tamoxifen The persistence of E, responsiveness of T,/Pr, cells was also confirmed by determining the antiproliferative effect of the antiestrogen TAM and its reversibility by E,. For the 5-day experiments, the cultures were exposed continuously to TAM and TAM plus E, and the nontreated cultures constituted the control set. In this short-term experiment, TAM induced a suppression of growth of approximately 62%, and E, was unable to reverse the antiproliferative effect of TAM, In
Estrogen dependence of mouse mammary tumor cells: Suto et al. % Estradiol metabolism (C-17 oxidation) 0.18
A
0.15
MMEC Vo pHO8T P-O.03 MMEC VI TllPrl Pr0.0001
t
% Estradiol metabolism 0.3 I
(C-2 hydroxylation)
a
1’
MMEC VI pHO8T P~O.0001
0.26
0.14
7
t 0.12
0.2
t 0.1 0.15 0.08
T
! 0.06
0.04 0.02 0I
I
0.1
0.05
I
MMEC
I
pH06T
8
I
0i
TllPfl
pHO6T
MMEC
T1/Pr1
% Estradiol metabolism (C-16alpha hydroxylation) C T
MMEC VI pHO3T PcO.0001 MMEC vs T1/Pr1 P~0.0001
MMEC
1
pHO0T
T1/Pr1
Figure 1 Relative extent of metabolic conversion of E2vi8 C-17 oxidation (A), C-2 hydroxyletion (6). and C-16~ hydroxylstion (C) pathways in MMEC (parental), pHO6T (ras transfected), and T,/Pr, (ras transformed) mammary epithelial cells.
the longer-term, IO-day cultures, after a S-day treatment with TAM, the cells were washed and maintained for the following 5 days either without any treatment (control) or in the medium supplemented with Er. As seen from the data presented in Figure 3, the growth inhibitory effect of TAM was effectively reversed by E 2. E#ect of indole-3-carbinol
In our recently completed in vivo study on C3H mice, 13C has been noted to alter E2 metabolism and suppress the growth of mammary tumors.20 To examine whether this agent is also effective in vitro on tumorigenically transformed mammary epithelial cells, T,/Pr, cells were exposed to the highest noncytotoxic level (50 PM) of 13C, and alteration in the extent of E2 metabolism and of anchorage-independent growth was examined. It is clear from the results presented in Table 3 that 13C exposure was effective in altering the metabolism of E2 specifically via the C-2 and C-l& hydroxylation pathways. Thus, while the extent of C-17 oxidation remained unaltered, C2 hydroxylation was upregulated at the expense of C-16o hydroxylation. To examine whether 13C can
alter the anchorage-independent growth of T,/Prl cells, the cultures were exposed continuously to 5, 50, and 500 PM 13C, and the number of colonies formed after 14 days was determined. As seen in Table 4, 13C induced a dose-associated experimental decrease in the number of anchorage-independent colonies, and 50 PM 13C was found to cause a 50% inhibition in the colony-forming efficiency of Ti/Pr, cells.
Discussion The responsiveness of mammary cancer to specific hormones plays an important role in adjuvant endocrine therapy.‘v2 The experiments conducted in the present study were designed to examine whether mammary epithelial cells that were tumorigenically transformed by the MS oncogene express persistent responsiveness to E2. Our previous studies have demonstrated that, independent of the type of initiating agent, the process of cell transformation is associated with alterations in E, metabolism in mammary epithelial cells. Oncogenic retrovirus, chemical carcinogens, and myc and rus oncogenes upregulate & C-&X hydroxylation at the exSteroids, 1992, vol. 57, June
265
Papers
pense of C-2 hydroxylation of Ez.r*-r6It is not known whether the fully transformed tumor cell phenotype retains its hormonal responsiveness. Acquisition of hormone independence for growth has been a frequently observed phenomenon during the progression of preneoplastically transformed mammary erithelial cells to a fully transformed mammary tumor. g*Similarly, a progressive loss of hormonal dependence has also been observed in rodent tumors.9,io,2~-23 An in vitro model mimicking in vivo endocrine responsiveness should provide an important experimental system wherein the molecular and cellular mechanisms responsible for the transition from hormone dependence to hormone independence can be systematically examined. The present study validates a reliable in vitro model derived from mouse mammary epithelial cell culture and demonstrates its utility as an experimental system for studying the endocri~ologic aspect of mammary cell transformation. Induction of hyperproliferation is clearly evident as an early occurring change in preneoplastically transformed pHO6T cells; this alteration persists in the tumor cell phenotype T,/Pr, . This proliferative change is manifested as a reduction in population doubling time 2 5 Cell number (XiO’f ‘P \ 2
*-** *-*** ,-****
1
P=O.O043 P~O.QOOl P~0.0001
.* 1.5
~ l
_s
1
0.5
a
-10
Concentration
20
of estradiol @oglMI)
HlThymidine Uptake(cpmXlO*) R ;-a. *-•**
P=O.O043 P~o.oo5t
-
I
l **
*t*t
15
:
-_
!
!
IO
1
i i
I
5
J..___ _:
Conlrol
-6
Concentration
-6
of estradiol (IogfMlf
Figure 2 Mitogenic effect of estradiol on res-transformed T,/Pr, cells meintained in the absence of serum and phenol red.
266
Steroids, 1992, vol. 53, June
3 $M_nufnber (X10? TAM(Day 2.5
10)
VS TAM+EP
P.O.0002
i
2-
1.5
‘.
/I 11
0.5 t
0
__-_-+_,
L.-...._L TAM fDey
10-6M 5)
TAM lO+M
TAM*E2 (Day
10“
M
10)
Figure3 Reversibility of the antiproliferative effect oftamoxifen by E2 in res-transformed T,/Pr, cells.
and acquisition of anchorage-independent growth. These results extend and confirm our previous observations, 16~17 and demonstrate the validity of hyperproliferation and anchorage-independent growth as specific in vitro markers for tumorigenic transformation. The endocrine responsiveness of mammary epithelial cells was examined by determining the relative extent of metabolism of E, via the commonly operative metabolic pathways of C-17 oxidation, C-2 hydroxylation, and C-~&J hydroxylation. It was interesting to note that in response to elevated expression of the MS oncogene, there was a specific increase in E, metabolism via the C-l& hydroxylation pathway but not via the C-2 hydroxylation pathway. This type of alteration is consistent with that reported in previous in vivo studies,1’*19*24 and also extends our recent in vitro studies r5*i6*i8 establishing the significance of the altered meiabolism of E2 as an endocrine biomarker of mammary cell transformation. Further confirmation of the estrogen-res~nsive nature of the rus-transformed cells is evident from the reversible inhibition of growth of these cells in phenol red-free medium. Phenol red, a commonly used pH indicator in tissue culture medium, has been noted for its estrogenic property. *KBIt was interesting to observe that the addition of phenol red resulted in a substantial increase in growth of cells maintained in the absence of this agent. The well-known antiestrogen, TAM, also exerted a growth inhibitor effect of T,fPr, cells. This inhibitory effect was not reversible by E, in cultures of 5 days’ duration, while in long-term cultures of 10days’ duration, Ez at 10e6 M and lo-* M doses induced increased cell growth, and thus was able to reverse the inhibitory effect of TAM. This preferential reversibility of TAM in long-term cultures but not in short-term cultures is difficult to interpret at this time. However, pleiotropic effects of antiestrogens are noted in human carcinoma-derived MCF-7 cell line.25*26 Depending on the duration of E, depletion, TAM has been noted to
Estrogen dependence of mouse mammary tumor cells: Suto et al. TI-
3 Effectof indoled-carbinol on estradiol metabolism in ras oncogana-transformed T,/Pr, calls Percentage of astradiol metabolisma
Treatment
C-17 oxidation
C-2 hydroxylation
C-l 6u hydroxylation
None l3C (50 UM) P
0.125 +- 0.039 0.113 k 0.055 n.s.
0.251 ” 0.033 0.640 ‘- 0.028
In vivo studies of spontaneous and/or ~~~inogeninduced rodent mammary can~e~,3~d~~~9-11 however, provide little ~ppo~unity for a mechanistic approach to analyze direct response of the target cells to initiators and rn~~~tors of neoplastic trausformation~ As an alternative approach, in vitro models developed from rn~rn~ epithe~al tissue have been used to identify molecular, metabolic, and cellular markers that are pe~nrbed due to initiation, and are susceptible to modutation by prototypic ~hemoprev~~tive ~ents,1z-~8 Recently we demonstrated that mammary epitheli~ cells from the low cancer risk BALBlc mouse can be tumorigenically t~~sfo~ed when transfected with ras and myc o~~ogenes.~~,~?Prior to the expression of fully transformed tumor cefl phenotypes the oncogenetransfected cells exhibit persistent expression of ras and myc t~~sc~pts, upre~ation of E, biotransfo~a~ tion via the C-160! hydroxylatio~ pathway, increased frequency of ancho~ge-independent growth, and a high incidence of hy~~lasti~ ~~t~owths from transplants. 16~17 Since all these end points precede the emer-
Estrogen de~e~de~~e of mouse mammae Table 1
Morphologic characteristics of mammary epithelial cells Origin
Cell line MMEC
tumor celfs: Suio et al,
MorphologV
Mammary tissue from virgin, female BALE/c mouse
Epithelioid colonies, polygonal cells. Form ‘normal’
ducts in perenchyma-free mammary fat pads. pHO6-f
MMEC transfected with the ras oncogene
T,/Pr,
Primary tumor from pH66T
In vitro morphology similer to MMEC. Form hyperplastic ducts in m8mm8~ fat pads. In vitro morphology similar to MMEC. Form rapidly growing tumors in mammary fat pads and at subcutaneous sites.
@Summarized from refs. 16 8nd 17.
gence of tumors, they can be considered as intermediate biomarkers for preneoplastic t~sfo~ation. In the present study, mammary epithelial cells derived from the BALBic mouse strain are used to induce tumorigenic transformation by the rus oncogene. The rus-transfected, tumorigenic cell is further characterized in vitro for the persistence of endocrine responsiveness and of tumorigenic phenotype. Furthermore, these perturbed biomarkers are used as specific end points to examine the modulatory infhrence of indole3-carbmol WC), a naturally occurring, potentially chemopreventive agent. Experimental
Ceil lines and chemicals The parental mouse mammary epithelial celi line (MMEC) and the rar oncogene transfectants were developed in the Breast Cancer Research Laboratory.i6 The characteristics of the cell lines MMEC (parental), pHO6T (rus transfected), and T,Pr, (tumor cell line derived from pHO6T) are provided in Table 1. Primary tumors from transplanted pHO6T cells were surgically excised, finely minced, and digested by incubation in a 1: 1 v/v mixture of collagenase-hyaluronidase according to published procedures. 15-17The cultures were plated in RJO-mmtissue culture plates and maintained in a humidified atmosphere of 95% air : 5% CO2 at 37 C using the DMEIF-12 medium (see below). The epithelioid colonies that grew were trypsinized and expanded in 25-cm2 flasks using the medium supplemented with 10% fetal bovine serum (FBS), These were designated as T,/Pr, cell line. The T,/Pr, cells at passage 80-90 were used for the experiments. Routinely, all the cell lines were maintained in Dulbecco’s Modified Eagle’s Medium F-12 (DMEJF-12, Sigma Chemical Co., St. Louis, MO, USA) supplemented with 10% FBS (heat inactivated), 4 mM L-glutamine, 100 U/ml penicillin, 100 pglml s~eptomycin, 100 ~glml gentamicin, and 5 pg/ml insulin (Eli Lilly Inc., Indianapolis, IN, USA), with phenol red as pH indicator. Estradiol, tamoxifen (TAM), and 13Cwere obtained from Sigma Chemical Co., and 13Hlthymidine (specitic activity, 40 to 60 Ci/mmol) was obtained from DuPont-NEN, Beverly, MA, USA. The concentrations of stock solutions of E2 and TAM were 1 x 1W* M, and of 13C were 5 x 10-l M.
by sequential reduction of FBS levels to 7.5%, 5.0%, 2.5%, and 1.0%. The cells that survived this selection procedure were evidently capable of growth in reduced levels of serumassociated endocrine factors, including estrogens. For the experiments on estrogen responsiveness, the stock culture was maintained in medium supplemented with 1% serum and 8.63 mg/L phenol red. Cell growth and proliferative activity were determined in serum-free condition with or without exogenous phenol red and Er .
~rawth kineti&s The cells were seeded at an initial density of 1 x 10s cells into T-25 flasks. After an attachment period of 18to 20 hours, the cultures were exposed to the test compounds for 2 to 5 days, depending on the specific experiment. At the end of this exposure, cell number and viability were determined by hemocytometer counts after staining the cells with trypan blue. The data were generated from at least six repiicate experiments.
~~H]~~ymidi~e uptake To determine the extent of rH]thymidine incorporation, the cells seeded at a density of 1.0 X 10scells were treated with noncytotoxic doses of estradiol and were exposed for the last 24 hours of culture to rH]thymidine (5 c~Ci/ml).The cultures were washed twice with cold phosphate-buffered saline to remove the unincorporated thymidine and lysed by exposure to 1 ml 1 N NaOH and 1% sodium dodecyl sulfate made up in phosp~te-bread saline. A total of 500 ~1 of the cellular lysate was then used to determine the ceUular uptake of 13Hlthymidine by liquid scintillation.13J4
Anchorage-independent
growth
To each well of a six-well plate was added 2 ml of the medium containing 0.6% agar (D&o, Nobel Co., Lincoln, NE, USA) to prepare the basal layer of agar. After an 18- to 20-hour incubation for soli~~tion of this layer, 2 ml of the medium containing 0.33% agar, 10% FBS, and 1 x 10’ dispersed cells was overlaid in each well. The plates were incubated for 14 days at 37 C in a humidified atmosphere of 5% CO* : 95% air, and the number of colonies ~40 pm formed was determined. ‘w’
Estrogen metabolism Adaptation of T&Pr, cells for growth in serumfree condition The effect of estrogenic agents was examined on the cells that were initiallyadapted to grow in serum-depletedmedium
The extent of e&radio1 rne~~~srn in the cells via C-17 oxidation, C-2 hydroxylation, and C-16~ hydroxylation pathways was determined by a radiometric assay. 1r*16$r This assay measures 3H exchange from stereospecifically labeled Er to form
Steroids, 1992, vol. 57, June
262
Papers
Table 2
Induction of hyperproliferation
and acquisition
of anchorage-independent
growth by ras oncogene
in mammary
epitheiial
cells Cell line
Population doubling timea
MMEC (parental) pHOST (ras transfected) T,/Pr, (ras transformed]
Number
27.8 rt 2.3c 22.0 it 1.8e 16.7 t 1.79
of anchorage-independent
coloniesb
3.7 zk 1.2d 150 t 28’ 335 2 46h
a Calculated from the linear portions of the growth curves. Mean 2 SD, n = 6. b Tridimensional colonies at day 14 per lo4 ceils plated in 0.33% agar. Mean rt SD, n = 18. c-e P = 0.004; c-9P = 0.001; d-fP < 0.0001; and *h P < 0.00001.
3H20. The amount of )HzO formed provides an indirect assessment of conversion of E2 to estrone (Et), and subsequently to Z-hydroxyestrone (2-OHE,) or to lfk-hydroxyestrone (ItSOHEl) in a stoichiometric manner.t9 The cells were incubated with [ 17-‘H]Er , [2-3HlE,, or [16&H]E2 (5 x lo6 dpm; specific activity, approximately 20 Ci/mmol) for 48 hours. Aliquots (500 ~1) of the medium were diluted to 3.5 ml with water and lyophilized, and sublimed water was counted in a liquid scintillation counter for ‘I&O formation. The extent of metabolism was corrected for nonspecific 3H exchange obtained from the incubations without the cells.
Statistical
analysis
The data were analyzed for their statistical significance by the two-sample t test where appropriate. Probability values of co.05 were considered significant.
Results Growth characteristics The initial experiments were designed to determine growth characteristics of the parental cell line MMEC,
the rus oncogene-transfected pHO6T, and the tumorigenie phenotype TJPr, by examining the alterations in population doubling times and the ability to undergo ancho~ge-inde~ndent growth. Population doubling times were found to decrease by approximately 21% and 40% in pHO6T and T,/Pr, cells, respectively, relative to that seen in MMEC. This decrease signifies enhancement of cell proliferative activity in the mammary epithelial cells that are expressing the transfected rus oncogene. Corresponding to the increased cell proliferative activity, pHO6T and T,/Pr, cells also exhibited a substantial increase in ~cho~ge-independent growth as evidenced by greater number of tridimensional colonies formed when suspended in semisolid medium containing 0.33% agar (Table 2). Estradiol responsiveness Persistence of the response of mammary epithelial cells to E, was evaluated by measuring the relative extent of E, metabolism via the C-17 oxidation, C-2 hydroxylation, and C-l& hydroxylation pathways. In general, estradiol metabolism was elevated in pHO6T and T,/ 264
Steroids,
1992, vol.
57, June
Pr, cells. The data presented in Figure 1 demonstrate that relative extent of C-17 oxidation of estradiol was elevated approximately two-fold in pHO6T and threefold in T,/Pr, cells relative to that in MMEC. The C-2 hydroxylation pathway was found to be downregulated in the pH06T cells but not in the T,/Pr, cells. It was interesting to note that the relative extent of estradiol metabolism via the C-I6a hydroxylation pathway exhibited a three-fold increase in ras-transfected cells and a 43-fold increase in rus-t~nsfo~ed cells. To further confirm the estrogen responsiveness, stock T,/Pr, cells were adapted to grow in serum-depleted (1% serum) medium containing phenol red. These cells routinely exhibited approximately 14-to 16fold increases in cell number within 5 days of culture. In contrast, cells grown in 1% serum without the estrogenie agent, phenol red, exhibited only a four- to fivefold increase in cell number. Thus, deletion of phenol red brought about a 73% decrease in growth of T,/ Pr, cells. A similar response to phenol red was also detected in cultures maintained in serum-free condition. As expected, in serum-free and phenol red-free condition T,/Pr, cells exhibited a nine-fold decrease in cell number at day 5 postseeding relative to the control (1% serum, phenol red-free medium). The addition of phenol red resulted in a 1Cfold increase in celI number. Similar to phenol red, E2 at a dose conventiona~y used in cell culture studies was also able to reverse the growth suppression caused by the deletion of serum and phenol red, as evidenced by an increase in cell number as well as in [3Hlthymidine uptake (Figure 2a,b). Thus, induction of growth by the estrogenic agent phenol red and by the natural estrogen, E2, provides strong evidence that T,/Pr, cells retain estrogen responsiveness. Effect of tamoxifen The persistence of E, responsiveness of T,/Pr, cells was also confirmed by determining the antiproliferative effect of the antiestrogen TAM and its reversibility by E,. For the 5-day experiments, the cultures were exposed continuously to TAM and TAM plus E, and the nontreated cultures constituted the control set. In this short-term experiment, TAM induced a suppression of growth of approximately 62%, and E, was unable to reverse the antiproliferative effect of TAM, In
Estrogen dependence of mouse mammary tumor cells: Suto et al. % Estradiol metabolism (C-17 oxidation) 0.18
A
0.15
MMEC Vo pHO8T P-O.03 MMEC VI TllPrl Pr0.0001
t
% Estradiol metabolism 0.3 I
(C-2 hydroxylation)
a
1’
MMEC VI pHO8T P~O.0001
0.26
0.14
7
t 0.12
0.2
t 0.1 0.15 0.08
T
! 0.06
0.04 0.02 0I
I
0.1
0.05
I
MMEC
I
pH06T
8
I
0i
TllPfl
pHO6T
MMEC
T1/Pr1
% Estradiol metabolism (C-16alpha hydroxylation) C T
MMEC VI pHO3T PcO.0001 MMEC vs T1/Pr1 P~0.0001
MMEC
1
pHO0T
T1/Pr1
Figure 1 Relative extent of metabolic conversion of E2vi8 C-17 oxidation (A), C-2 hydroxyletion (6). and C-16~ hydroxylstion (C) pathways in MMEC (parental), pHO6T (ras transfected), and T,/Pr, (ras transformed) mammary epithelial cells.
the longer-term, IO-day cultures, after a S-day treatment with TAM, the cells were washed and maintained for the following 5 days either without any treatment (control) or in the medium supplemented with Er. As seen from the data presented in Figure 3, the growth inhibitory effect of TAM was effectively reversed by E 2. E#ect of indole-3-carbinol
In our recently completed in vivo study on C3H mice, 13C has been noted to alter E2 metabolism and suppress the growth of mammary tumors.20 To examine whether this agent is also effective in vitro on tumorigenically transformed mammary epithelial cells, T,/Pr, cells were exposed to the highest noncytotoxic level (50 PM) of 13C, and alteration in the extent of E2 metabolism and of anchorage-independent growth was examined. It is clear from the results presented in Table 3 that 13C exposure was effective in altering the metabolism of E2 specifically via the C-2 and C-l& hydroxylation pathways. Thus, while the extent of C-17 oxidation remained unaltered, C2 hydroxylation was upregulated at the expense of C-16o hydroxylation. To examine whether 13C can
alter the anchorage-independent growth of T,/Prl cells, the cultures were exposed continuously to 5, 50, and 500 PM 13C, and the number of colonies formed after 14 days was determined. As seen in Table 4, 13C induced a dose-associated experimental decrease in the number of anchorage-independent colonies, and 50 PM 13C was found to cause a 50% inhibition in the colony-forming efficiency of Ti/Pr, cells.
Discussion The responsiveness of mammary cancer to specific hormones plays an important role in adjuvant endocrine therapy.‘v2 The experiments conducted in the present study were designed to examine whether mammary epithelial cells that were tumorigenically transformed by the MS oncogene express persistent responsiveness to E2. Our previous studies have demonstrated that, independent of the type of initiating agent, the process of cell transformation is associated with alterations in E, metabolism in mammary epithelial cells. Oncogenic retrovirus, chemical carcinogens, and myc and rus oncogenes upregulate & C-&X hydroxylation at the exSteroids, 1992, vol. 57, June
265
Papers
pense of C-2 hydroxylation of Ez.r*-r6It is not known whether the fully transformed tumor cell phenotype retains its hormonal responsiveness. Acquisition of hormone independence for growth has been a frequently observed phenomenon during the progression of preneoplastically transformed mammary erithelial cells to a fully transformed mammary tumor. g*Similarly, a progressive loss of hormonal dependence has also been observed in rodent tumors.9,io,2~-23 An in vitro model mimicking in vivo endocrine responsiveness should provide an important experimental system wherein the molecular and cellular mechanisms responsible for the transition from hormone dependence to hormone independence can be systematically examined. The present study validates a reliable in vitro model derived from mouse mammary epithelial cell culture and demonstrates its utility as an experimental system for studying the endocri~ologic aspect of mammary cell transformation. Induction of hyperproliferation is clearly evident as an early occurring change in preneoplastically transformed pHO6T cells; this alteration persists in the tumor cell phenotype T,/Pr, . This proliferative change is manifested as a reduction in population doubling time 2 5 Cell number (XiO’f ‘P \ 2
*-** *-*** ,-****
1
P=O.O043 P~O.QOOl P~0.0001
.* 1.5
~ l
_s
1
0.5
a
-10
Concentration
20
of estradiol @oglMI)
HlThymidine Uptake(cpmXlO*) R ;-a. *-•**
P=O.O043 P~o.oo5t
-
I
l **
*t*t
15
:
-_
!
!
IO
1
i i
I
5
J..___ _:
Conlrol
-6
Concentration
-6
of estradiol (IogfMlf
Figure 2 Mitogenic effect of estradiol on res-transformed T,/Pr, cells meintained in the absence of serum and phenol red.
266
Steroids, 1992, vol. 53, June
3 $M_nufnber (X10? TAM(Day 2.5
10)
VS TAM+EP
P.O.0002
i
2-
1.5
‘.
/I 11
0.5 t
0
__-_-+_,
L.-...._L TAM fDey
10-6M 5)
TAM lO+M
TAM*E2 (Day
10“
M
10)
Figure3 Reversibility of the antiproliferative effect oftamoxifen by E2 in res-transformed T,/Pr, cells.
and acquisition of anchorage-independent growth. These results extend and confirm our previous observations, 16~17 and demonstrate the validity of hyperproliferation and anchorage-independent growth as specific in vitro markers for tumorigenic transformation. The endocrine responsiveness of mammary epithelial cells was examined by determining the relative extent of metabolism of E, via the commonly operative metabolic pathways of C-17 oxidation, C-2 hydroxylation, and C-~&J hydroxylation. It was interesting to note that in response to elevated expression of the MS oncogene, there was a specific increase in E, metabolism via the C-l& hydroxylation pathway but not via the C-2 hydroxylation pathway. This type of alteration is consistent with that reported in previous in vivo studies,1’*19*24 and also extends our recent in vitro studies r5*i6*i8 establishing the significance of the altered meiabolism of E2 as an endocrine biomarker of mammary cell transformation. Further confirmation of the estrogen-res~nsive nature of the rus-transformed cells is evident from the reversible inhibition of growth of these cells in phenol red-free medium. Phenol red, a commonly used pH indicator in tissue culture medium, has been noted for its estrogenic property. *KBIt was interesting to observe that the addition of phenol red resulted in a substantial increase in growth of cells maintained in the absence of this agent. The well-known antiestrogen, TAM, also exerted a growth inhibitor effect of T,fPr, cells. This inhibitory effect was not reversible by E, in cultures of 5 days’ duration, while in long-term cultures of 10days’ duration, Ez at 10e6 M and lo-* M doses induced increased cell growth, and thus was able to reverse the inhibitory effect of TAM. This preferential reversibility of TAM in long-term cultures but not in short-term cultures is difficult to interpret at this time. However, pleiotropic effects of antiestrogens are noted in human carcinoma-derived MCF-7 cell line.25*26 Depending on the duration of E, depletion, TAM has been noted to
Estrogen dependence of mouse mammary tumor cells: Suto et al. TI-
3 Effectof indoled-carbinol on estradiol metabolism in ras oncogana-transformed T,/Pr, calls Percentage of astradiol metabolisma
Treatment
C-17 oxidation
C-2 hydroxylation
C-l 6u hydroxylation
None l3C (50 UM) P
0.125 +- 0.039 0.113 k 0.055 n.s.
0.251 ” 0.033 0.640 ‘- 0.028
