Estrogen mitogenic action. I. Demonstration of estrogen-dependent MTW9/PL2 carcinogen-induced rat mammary tumor cell growth in serum-supplemented culture and technical implications.

The MTW9/PL cell line was established by our laboratory in culture from the carcinogen-induced hormone-responsive MT-W9A rat mammary tumor of a Wistar-Furth (W/Fu) rat. This tumor formed estrogen, androgen, and progesterone responsive tumors in W/Fu rats (Sirbasku, D. A., Cancer Res. 38:1154-1165; 1978). It was later used to derive the MTW9/PL2 cell population which was also estrogen-responsive in vivo (Danielpour, D., et al., In Vitro Cell. Dev. Biol. 24:42-52; 1988). In the study presented here, we describe serum-supplemented culture conditions in which the MTW9/PL2 cells demonstrate > or = 80-fold steroid hormone growth responses. All sera used were steroid hormone-depleted by charcoal-dextran treatment at 34 degrees C. The studies were done with horse serum as well as serum from other mammalian species. The growth of the MTW9/PL2 cells was biphasic in response to hormone-depleted serum. Concentrations of < or = 5% (v/v) promoted optimum growth. Above this concentration, serum was inhibitory. Concentrations > or = 40% (v/v) inhibited growth altogether. Addition of 1.0 x 10(-13)-1.0 x 10(-8) M 17beta,-estradiol (E2) reversed the inhibition completely. At 1.0 x 10(-8) M, estrone, estriol and diethylstilbestrol promoted growth as well as E2. Testosterone and dihydrotestosterone promoted growth only at > or = 10(-7) M. Progesterone was effective only at > or = 10(-6) M. Cortisol was ineffective. Labeled-hormone-binding analysis and Western immunoblotting documented that MTW9/PL2 cells had estrogen and progesterone receptors but not androgen or cortisol receptors. Estrogen treatment of MTW9/PL2 cells induced a concentration and time dependent increase in progesterone receptors. We conclude (1) the MTW9/PL2 population is the first highly steroid hormone-responsive rat mammary tumor cell line to be established in culture from a carcinogen-induced tumor, and (2) sera from a number of species including horse, rat and human contain an inhibitor which mediates estrogen sensitive MTW9/PL2 cell growth in culture.

Estrogen mitogenic action. III. is phenol red a "red herring"?

The reported estrogenic action of phenol red and/or its lipophilic contaminants has led to the widespread use of indicator-free culture medium to conduct endocrine studies in vitro. Because we have recently developed methods to measure large-magnitude estrogen effects in the tissue culture medium containing phenol red, we concluded that the indicator issue required further evaluation. To do this, we selected nine estrogen receptor positive (ER+) cell lines representing four target tissues and three species. We investigated phenol red using five different experimental protocols. First, 17beta-estradiol (E2) responsive growth of all nine ER+ cells lines was compared in the medium with and without the indicator. Second, using representative lines we asked if phenol red was mitogenic in the indicator-free medium. The dose-response effects of phenol red were compared directly to those of E2. Third, we asked if tamoxifen-inhibited growth equally in phenol red-containing and indicator-free medium. This study was based on a report indicating that antiestrogen effects should be seen only in phenol red-containing medium. Fourth, we asked if phenol red displaced the binding of 3H-E2 using ERK intact human breast cancer cells. Fifth, we compared E2 and phenol red as inducers of the progesterone receptor using a human breast cancer cell line. All the experiments presented in this report support the conclusion that the concentration of phenol red contaminants in a standard culture medium available today is not sufficient to cause estrogenic effects. In brief, our studies indicate that the real issue of how to demonstrate estrogenic effects in culture resides elsewhere than phenol red. We have found that the demonstration of sex steroid hormone-mitogenic effects in culture depends upon conditions that maximize the effects of a serum-borne inhibitor(s). When the effects of the inhibitor are optimized, the presence or absence of phenol red makes no everyday difference to the demonstration of estrogen mitogenic effects with several target cell types from diverse species.

Estrogen mitogenic action. ii. negative regulation of the steroid hormone-responsive growth of cell lines derived from human and rodent target tissue tumors and conceptual implications.

In an accompanying report (Moreno-Cuevas, J. E.; Sirbasku, D. A., In Vitro Cell. Dev. Biol.; 2000), we demonstrated 80-fold estrogen mitogenic effects with MTW9/PL2 rat mammary tumor cells in cultures supplemented with charcoal-dextran-treated serum. All sera tested contained an estrogen reversible inhibitor(s). The purpose of this report is to extend those observations to additional sex steroid-responsive human and rodent cell lines. Every line tested showed a biphasic response to hormone-depleted serum. Concentrations of < or = 10% (v/v) promoted substantive growth. At higher concentrations, serum was progressively inhibitory. With estrogen receptor-positive (ER+) human breast cancer cells, rat pituitary tumor cells, and Syrian hamster kidney tumor cells, 50% (v/v) serum caused significant inhibition, which was reversed by very low physiologic concentrations of estrogens. This same pattern was observed with the steroid hormone-responsive LNCaP human prostatic carcinoma cells. Because steroid hormone mitogenic effects are now easily demonstrable using our new methods, the identification of positive results has nullified our original endocrine estromedin hypothesis. We also evaluated autocrine/paracrine growth factor models of estrogen-responsive growth. We asked if insulin-like growth factors I and II, insulin, transforming growth factor alpha, or epidermal growth factor substituted for the positive effects of estrogens. Growth factors did not reverse the serum-caused inhibition. We asked also if transforming growth factor beta (TGFP) substituted for the serum-borne inhibitor. TGFbeta did not substitute. Altogether, our results are most consistent with the concept of a unique serum-borne inhibitor as has been proposed in the estrocolyone model. However, the aspect of the estrocolyone model related to steroid hormone mechanism of action requires more evaluation. The effects of sex steroids at picomolar concentrations may reflect mediation via inhibitor "activated" intracellular signaling pathways.

Apotransferrin stimulation of thyroid hormone dependent rat pituitary tumor cell growth in serum-free chemically defined medium: role of FE(III) chelation.

Triiodothyronine (T3) dependent growth of GH1 rat pituitary tumor cells in serum-free defined culture requires apotransferrin (apoTf) (Sirbasku et al.: Mol. Cell. Endocrinol., 77:C47-C55, 1991). Diferric transferrin (2Fe.Tf) also is necessary as an iron source (Eby et al.: Anal. Biochem., 203:317-325, 1992). Further, T3 dependence is prevented by soluble Fe(III) addition to the medium (Sato et al.: In Vitro Cell. Dev. Biol., 27A:599-602, 1991). While our data suggested that apoTf caused growth by chelation of Fe(III), direct evidence was required. We used urea polyacrylamide gel electrophoresis along with autoradiography and Western immunoblotting to measure the Fe(III) content of growing GH1 cell cultures and identify the apoTf, mono-metal transferrins and 2Fe.Tf present. We found that apoTf per se did not cause growth but instead chelated inhibitory levels of Fe(III). In fact, apoTf need not be present at all provided that Fe(III) is reduced to < or = 0.6 microM. In addition, other protein and non-protein Fe(III) chelators were shown to be as effective as apoTf. Here, we report that pituitary cells are completely inhibited by > or = 1.2 microM Fe(III), which are concentrations which might be expected in many culture media and usually are not thought to influence growth. The high sensitivity of pituitary cells to Fe(III) suggests further study to determine what cellular functions are affected and how they interfere with thyroid hormone dependence.

Preparation of iron-deficient tissue culture medium by deferoxamine-sepharose treatment and application to the differential actions of apotransferrin and diferric transferrin.

We have shown that triiodothyronine-dependent GH1 rat pituitary cell growth in serum-free defined culture required apotransferrin (apoTf) (D. A. Sirbasku, et al., Biochemistry 30, 295-304, 7466-7477, 1991). These studies were done in "low-Fe" medium without Fe(III)/Fe(II) salts. Nonetheless, significant concentrations of iron may have been contributed by other components, making this medium unsuitable for study of the differential effects of apoTf and diferric transferrin (2Fe.Tf). Measuring residual iron in culture medium has been troublesome because the most sensitive method (i.e., atomic absorption) detected levels only in excess of 10 ng/ml and did not distinguish between the forms of iron present. To estimate the Fe(III) available to bind to apoTf, we developed a more sensitive and specific method. Urea-polyacrylamide gel electrophoresis (PAGE) separates apoTf, the two monoferric transferrins, and 2Fe.Tf. [125I]apoTf was incubated with medium, or components, and the formation of [125I]-2Fe.Tf was monitored by urea-PAGE/autoradiography. By this method, the concentration of Fe(III) in low-Fe medium was estimated at 8.4 to 20 ng/ml and the sources were identified. We next sought to remove the Fe(III). Standard chelators were ineffective or cytotoxic. In contrast, an affinity method with deferoxamine-Sepharose depleted greater than or equal to 90% of the Fe(III). In this medium, apoTf and 2Fe.Tf showed differential effects with GH1 cells and with MCF-7, MTW9/PL2, an MDCK cells. With the methods described here, the effects of apoTf and 2Fe.Tf on growth can be studied separately.

Apotransferrins from several species promote thyroid hormone-dependent rat pituitary tumor cell growth in iron-restricted serum-free defined culture.

Previously, we have studied thyroid hormone-dependent growth of GH1 rat pituitary tumor cells in iron-restricted serum-free defined medium (Sirbasku, D.A., et al. (1991) Biochemistry 30, 295-304, 7466-7477). Proliferation was promoted by triiodothyronine (T3) and any of seven forms of horse serum-derived apotransferrin (apoTf). In this report, we have asked if apoTfs from other species also acted as thyromedins and if other metal ion chelators served this role. To address these issues, three thyromedins were isolated from human serum and identified as apoTf. Fe3+ depletion, and assay in low-Fe medium, gave ED50s of 1.4-1.7 nM. Fe3+ saturation abolished their activities in high-Fe medium. To ask if apoTf was the major thyromedin in human serum, hormone-depleted preparations were iron saturated and shown to no longer support T3-dependent GH1 cell growth. Next, commercially prepared human, rat, horse, dog, rabbit, guinea pig and mouse apoTfs were shown to be as active under iron-restricted conditions as those isolated from human serum. Bovine apoTf and colostrum lactoferrin were greater than 100-fold less active; human milk apo-lactoferrin and apo-ovotransferrins were inactive. Transferrins which displayed thyromedin activity blocked the binding of 125I-rat 2Fe.Tf to GH1 cell receptors while those without thyromedin activity were ineffective. Finally, the metal ion chelators EDTA, citrate and deferoxamine did not show thyromedin activity indicating that apoTfs uniquely were able to promote T3-dependent cell growth in defined culture.

Thyroid hormone and apotransferrin regulation of growth hormone secretion by GH1 rat pituitary tumor cells in iron restricted serum-free defined medium.

Growth hormone (GH) production by GH1 rat pituitary tumor cells in iron restricted serum-free defined medium requires apotransferrin (apoTf) and triiodothyronine (T3). As measured by radioimmunoassay, apoTf plus T3 induced GH levels 2 to 4-fold above controls. Deletion of either apoTf or T3 arrested GH secretion. ApoTf/T3 defined medium regulated GH production as effectively as whole serum. Because glucocorticoids enhance GH secretion in serum containing cultures, the effects of dexamethasone were evaluated in apoTf/T3 defined medium. The steroid hormone showed no enhancing effects unless the cells were exposed to serum prior to incubation in apoTf/T3 defined medium. Even under these conditions, the response to dexamethasone remained T3 dependent. These observations indicate that a yet to be characterized serum factor(s), other than apoTf, regulates the response to the steroid hormone. This is the first report of thyroid hormone regulation of GH secretion by rat pituitary tumor cells under completely serum-free chemically defined conditions.

Thyroid hormone dependent pituitary tumor cell growth in serum-free chemically defined culture. A new regulatory role for apotransferrin.

Thyroid hormone dependent GH1 rat pituitary tumor cell growth in serum-free chemically defined medium required a serum-derived mediator (i.e., thyromedin) which was identified as transferrin [Sirbasku, D.A., Stewart, B.H., Pakala, R., Eby, J.E., Sato, H., & Roscoe, J.M. (1990) Biochemistry 30, 295-304]. The transferrin isolated was consistent with the equine R or D variants and was biologically active only as apotransferrin (apoTf). To determine if other variants of horse transferrin also were thyromedins, a purification was developed which yielded seven separate forms. Initially, only four of these had activity when assayed in standard "iron salts containing" medium (ED50 values of 290-1160 nM). To further assess activity, the iron contents of all seven were altered either by saturation with ferric ammonium citrate or by citrate/acid depletion of the metal ion. Thereafter, potencies were compared in "iron salts containing" and "iron salts reduced" media. All seven variants proved to be active as apoTf. Bioassays in which apoTf was maximized showed ED50 values of 2.1-3.8 nM. Conversely, assays in which thyromedins were converted to Tf.2Fe showed no activity. Previously, the only known physiological function of apoTf was that of a carrier/detoxifier of iron; this study indicates a new role in hormone-dependent pituitary cell growth.

Control of cell death (apoptosis) by diethylstilbestrol in an estrogen-dependent kidney tumor.

The role of cell death as a determinant for tumor growth and regression was studied using an estrogen-dependent, transplantable kidney tumor designated H301. H301 cells were injected s.c. into diethylstilbestrol(DES)-treated male Syrian hamsters and developed solid tumors of 1-2 g within 2-3 weeks. Upon withdrawal of estrogen the tumors regressed by 80-90% within 4 days. Mitoses, necrotic areas and single-cell death indicated by small, condensed cell residues, were counted in hematoxylin and eosin stained histological sections of the tumors. Coincident with tumor regression after DES withdrawal, mitotic activity decreased by approximately 90%; the rate of single-cell death increased (by approximately 2-fold at its maximum). The incidence of necrotic areas was not affected by DES withdrawal. DES re-treatment resulted in reduction of single-cell death by 80% within 8 h. Mitotic activity increased within 24 h to the level observed before DES withdrawal. Again, the incidence of necrotic areas did not change. As a result, tumors re-grew to their previous size within 2 days after resumption of DES treatment. These results led to the following conclusions: (i) DES treatment inhibits and DES withdrawal enhances single-cell death of H301 tumor cells. (ii) Both this functional property and its morphology characterize single-cell death in the tumors as apoptosis. (iii) Estrogen-dependent cell death determines, in addition to mitosis and necrosis, the growth rate of H301 tumors. (iv) This experimental model may provide a useful tool to study the interaction of potential anti-tumor drugs with apoptosis in neoplasia.

Thyroid hormone regulation of rat pituitary tumor cell growth: a new role for apotransferrin as an autocrine thyromedin.

In the 40 years of transferrin research, no previous role for apotransferrin has been recognized other than to serve as a plasma carrier for dietary and storage iron. Our studies have revealed a new 'autocrine' growth role for this molecule as well as a possible new cell-cell bridge/CAM function. Certainly, these observations have opened many new areas of investigation both with regard to thyroid hormone action and the function of apotransferrin. In addition, there is now accessible the broader question of tissues other than pituitary which might utilize apotransferrin to regulate responsiveness to thyroid hormones.

Purification of an equine apotransferrin variant (thyromedin) essential for thyroid hormone dependent growth of GH1 rat pituitary tumor cells in chemically defined culture.

Pituitary tumor cells require thyroid hormones for growth in vivo [Sorrentino, J. M., Kirkland, W. L., & Sirbasku, D. A. (1976) J. Natl. Cancer Inst. 56, 1155-1158]. In vitro, GH1 rat pituitary tumor cells were studied in a serum-free defined medium (PCM-10) formulated with Ham's F12 and Dulbecco's modified Eagle's media (1:1, v/v) supplemented with 2.2 g/L sodium bicarbonate, 15 mM 4-(2-hydroxy-ethyl)-1-piperazineethanesulfonic acid (pH 7.2), 10 micrograms/mL human transferrin, 50 microM ethanolamine, 10 micrograms/mL insulin, 10 ng/mL selenous acid, 0.1 nM 3,5,3'-triiodothyronine (T3) and 500 micrograms/mL bovine serum albumin and in the same medium without T3 (PCM-0). The cells only grew in PCM-10 when low concentrations of horse serum were added. Attempts to replace the serum factor requirement with known growth factors and adhesion proteins were unsuccessful. The Mr 65,000-72,000 serum factor regulating T3-induced growth (thyromedin) was purified to homogeneity and identified as equine transferrin R and/or D by amino acid sequencing. The ED50 in PCM-10 was 17-40 micrograms/mL (260-620 nM) while in PCM-0 half-maximum growth was not achieved at 200 micrograms/mL. Concentrations of 75 micrograms/mL in PCM-10 caused 80% of serum-stimulated growth rate. Removal of iron from thyromedin, and assay in iron salts reduced PCM-10, increased the specific activity 110-270-fold to ED50 150 ng/mL (2.3 nM); at 1.0 micrograms/mL, growth in PCM-10 was 16-fold greater than in PCM-0. Iron saturation of thyromedin caused total loss of biological activity. We conclude that the horse transferrin variant isolated in this report is active as apotransferrin.

Human platelet-derived mitogens. I. Identification of insulinlike growth factors I and II by purification and N alpha amino acid sequence analysis.

Human platelet lysates contained potent mitogenic activities for MCF-7 human breast-cancer cells in serum-free-defined media. Because these activities were not replaced by known platelet mitogens, such as platelet-derived growth factor or transforming growth factor beta, we sought to identify the breast cancer cell mitogens by purification and N alpha amino-acid sequencing. Acetic acid extracts of outdated human platelets were concentrated by ammonium sulfate precipitation and fractionated on Sephadex G-50 and Bio-Gel P-10 columns in 0.5 mol/L acetic acid. Two major activities were resolved by molecular sieve methods and fractionated further by reverse-phase high-performance liquid chromatography (HPLC). Purifications (70,000 to 870,000-fold) were accomplished yielding mol wt 7,400 products that were homogeneous as determined by iodination, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and autoradiography. The factors were identified as insulinlike growth factor I (IGF-I) and II (IGF-II) and truncated IGF-I by N alpha amino acid microsequencing. In dose-response experiments, platelet-derived IGF-I and IGF-II promoted multiple divisions of the MCF-7 cells with ED50 values of 12 and 100 pg/mL, respectively. The specific activities and other bioassay characteristics of platelet-derived IGF-I and IGF-II were similar to those of recombinant-produced human growth factors. This is the first report of the purification of insulinlike growth factors from human platelet lysates.

Human platelet-derived mitogens. II. Subcellular localization of insulinlike growth factor I to the alpha-granule and release in response to thrombin.

Platelets contain mitogenic activities for MCF-7 human breast cancer cells when assayed under serum-free chemically defined conditions. Purification from outdated human platelets identified insulinlike growth factor I (IGF-I) as the most potent breast cancer cell mitogen in lysates (Karey KP, Sirbasku DA: see accompanying article, this issue). In this study the release and subcellular localization of IGF-I was investigated. Degranulation of platelets by thrombin treatment caused release of lysosomal enzymes (beta-glucuronidase and N-acetyl-D-glucosaminidase), alpha-granule proteins (beta-thromboglobulin and fibrinogen) as well as mitogenic activity for MCF-7 cells and IGF-I as measured by radioimmunoassay (RIA) and radioreceptor assay. Release of mitogenic activity and immunologically identified IGF-I was induced tenfold over controls by thrombin and was nearly complete as compared to platelets disrupted by repeated freezing and thawing. Disruption of platelets by nitrogen cavitation followed by separation of the organelles by sucrose density gradient sedimentation showed that IGF-I and mitogenic activity localized predominantly to fractions containing alpha-granules rather than soluble cellular components, lysosomes, or dense granules. The morphology of MCF-7 cells in serum-free medium supplemented with supernatants from thrombin-treated platelets also indicated the release of important cell-adhesion factors for human breast cancer cells.

Glutaraldehyde fixation increases retention of low molecular weight proteins (growth factors) transferred to nylon membranes for western blot analysis.

Western blotting of low molecular weight (Mr) acidic and basic isoelectric point (pI) proteins was studied to optimize detection sensitivities. Radioiodinated epidermal growth factor (EGF, Mr 6045, pI 4.4), transforming growth factor type alpha (TGF alpha, Mr 5623, pI 6.8), insulin-like growth factor (IGF-I, Mr 7649, pI 8.3), and basic fibroblast growth factor (bFGF, Mr 15,000-17,000, pI 9.6) all transferred with high efficiency (74.1 +/- 12.6%) to a positively charged nylon membrane. Sequential application of standard unoccupied site blocking, antibody incubation, and washing steps resulted in significant losses of all growth factors (46-98%). Basic FGF was retained best. Treatment of transfer membranes with 0.5% (v/v) glutaraldehyde prior to blocking and immunodetection increased the retention of the growth factors 1.5- to 12-fold over untreated controls. Without fixation, 100 ng of EGF, TGF alpha, and IGF-I were not detectable while 6.25-100 ng was identified on fixed membranes. The methods described were equally sensitive for detecting both acidic and basic pI proteins.

Identification and purification of truncated insulin-like growth factor I from porcine uterus. Evidence for high biological potency.

We report the completion of the purification of uterine-derived growth factors (UDGF) described previously by this laboratory [Ikeda, T., & Sirbasku, D.A. (1984) J. Biol. Chem. 259, 4049-4064]. During isolation, the mitogenic activity was monitored by using the human MCF-7 breast cancer cells in serum-free Ham's F12 and Dulbecco's modified Eagle's medium (1:1, v/v) containing 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.2), 200 micrograms/mL bovine serum albumin, and 10 micrograms/mL human transferrin. This medium sustained growth for several days in response to a single addition of growth factor. The isolation of UDGF began with acetic acid extraction followed by sulfopropyl-Sephadex chromatography, Bio-Gel P-10 molecular sieve fractionation, and a series of reverse-phase high-pressure liquid chromatography separations. Purifications [[(1.0-8.5) X 10(6)]-fold] of three mitogens (5-20 ng each) were achieved. The mitogens were shown by protein microsequencing to be DES 1----3 to DES 1----6 forms of insulin-like growth factor I (truncated IGF-I). An Mr estimated by 125I labeling, urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography was consistent with a DES 1----3(4) N alpha truncation. Immunoadsorption and radioimmunoassay confirmed immunological properties equivalent to IGF-I. Radioreceptor assays showed truncated IGF-I was functionally equivalent to recombinant IGF-I. The ED50 values of DES 1----3 IGF-I and recombinant IGF-I for MCF-7 cell growth were 0.8-6.0 and 30-150 pg/mL, respectively. With Balb/c 3T3 mouse fibroblasts, the ED50 of DES 1----3 IGF-I was 100 times lower than that of IGF-I. We conclude that the major acid-stable low-Mr mitogenic activities isolated from uterus are very potent forms of truncated IGF-I capable of stimulating growth of epithelial and mesenchymal cells.

Identification of a 15,000-molecular-weight form of immunoreactive transforming growth factor alpha in extracts of porcine pituitary.

Two different mitogenic activities were identified from extracts of porcine pituitary by using COMMA-D mouse mammary epithelial cells in a serum-free 3H-thymidine incorporation assay. Porcine pituitaries were extracted in phosphate-buffered saline (pH 7.4) and 25-80% (NH4)2SO4 pellets were dialyzed and chromatographed by using DEAE-Sepharose chromatography (pH 8.0), resulting in two peaks (I and II) of mitogenic activity. Peak I represented a recovery of 73% of the units of mitogenic activity present in crude extract of pituitary while only 1.25% of the activity was recovered in peak II. Peak I was further purified by using CM-Sephadex and heparin-Sepharose chromatographies and yielded a mitogen that was able to elicit one-half-maximal stimulation of 3H-thymidine incorporation by COMMA-D cells at 48 pg/ml. As expected with pituitary as the tissue source, peak I was confirmed to be basic fibroblast growth factor (bFGF) by using specific antibodies in enzyme-linked immunosorbent assay and Western immunoblotting procedures. Peak II was further purified by using chromatofocusing (pH 7.3-5.0), reverse-phase, and cation-exchange HPLCs. The mitogenic activity eluted at pH 6.3 from chromatofocusing, migrated as a 13-kDa molecule on gel filtration HPLC, and did not bind to heparin-Sepharose under conditions which bound fibroblast growth factors. The material purified from peak II and rat synthetic transforming growth factor alpha (TGF alpha) competed in a parallel fashion with 125I-epidermal growth factor for receptor binding with A431 human epidermal carcinoma cells. In addition, the mitogen purified from peak II showed a single immunoreactive band migrating at 15 kDa when specific antiserum against TGF alpha was used in a Western immunoblotting procedure. The data suggest that in addition to the well-documented presence of bFGF, normal adult porcine pituitaries contain a 15-kDa form of immunoreactive TGF alpha that binds to EGF receptors and is mitogenic for mammary epithelial cells.

Characterization of polyclonal antibodies that distinguish acidic and basic fibroblast growth factors by using western immunoblotting and enzyme-linked immunosorbent assays.

Rabbit polyclonal antibodies were raised against ovalbumin conjugates of purified bovine brain acidic fibroblast growth factor (aFGF) and a synthetic peptide containing the N alpha-terminal 1-24 amino acid sequence of bovine basic fibroblast growth factor (bFGF). These antibodies were used to specifically detect 1-ng quantities of aFGF and bFGF by using enzyme-linked immunosorbent assay (ELISA) and Western immunoblot procedures. Antibodies raised against aFGF recognized bovine brain aFGF and bovine recombinant aFGF but very poorly recognized recombinant bFGF or purified porcine or bovine pituitary bFGF with ELISA and Western immunoblot procedures. Antibodies raised against bFGF (1-24) recognized purified bovine, porcine, and recombinant human bFGF but only very poorly recognized aFGF with ELISA and Western immunoblot procedures. In vitro addition of anti-bFGF antibodies was able to partially neutralize bFGF-stimulated 3H-thymidine incorporation by COMMA-D mouse mammary epithelial cells while having no effect on aFGF or epidermal growth factor (EGF) stimulation. In vitro addition of anti-aFGF antibodies had no effect on bFGF- or EGF-stimulated 3H-thymidine incorporation, but surprisingly, had a potentiating effect on aFGF stimulation. Antibodies against aFGF immobilized on protein A-Sepharose were able to specifically and completely remove mitogenic activity from solutions containing aFGF but had no effect on removal of mitogenic activity from control solutions containing bFGF or EGF. Similarly, immobilized anti-bFGF antibodies completely removed mitogenic activity from solutions of bFGF, but not aFGF or EGF controls. These antibodies have been useful for the identification and characterization of growth factors from tissue and recombinant sources.

Rat pituitary tumor cells in serum-free culture. I. Selection of thyroid hormone-responsive and autonomous cells.

The growth of GH4C1, GH3, GH1, and GH3C15 rat pituitary tumor cell lines was studied in a serum-free medium (designated TRM-1) formulated with 1:1 (vol/vol) mixture of Ham's F12 nutrient mixture and Dulbecco's modified Eagle's medium (F12-DME) containing 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 50 micrograms/ml gentamicin supplemented with 10 micrograms/ml bovine insulin, 10 micrograms/ml human transferrin (Tf), 10 ng/ml selenous acid, 10 nM 3,5,3'-triiodothyronine (T3), 50 microM ethanolamine (Etn), and 500 micrograms/ml bovine serum albumin. Of the lines evaluated, only the GH1 failed to grow in TRM-1. Passage of the GH4C1 and GH3 lines from serum-containing medium into TRM-1 caused an initial selection resulting in cells that grew progressively at higher rates and finally were maintained indefinitely in TRM-1. These populations showed a requirement for supraphysiologic concentrations of T3 (1.0 to 10 nM). After adaptation of the GH4C1 line in TRM-1 for greater than or equal to 20 generations, removal of components gave a less complex mixture containing 15 mM HEPES, 50 micrograms/ml gentamicin, 10 micrograms/ml Tf, 10 nM T3, and 50 microM Etn (designated TRM-2) that supported serial passage of the cells. Under these conditions, thyroid hormone dependence was lost progressively. When T3 was removed from TRM-2 adapted cells, a third population was selected that no longer required thyroid hormones and was only slightly stimulated by T3. These studies demonstrated that the combination of serum-containing and serum-free conditions can be used to select pituitary cell populations that a) required both serum-factor(s) and T3 for optimum growth, b) required supraphysiologic concentrations of T3 without serum proteins other than Tf and albumin, and c) were completely autonomous in that they proliferated in medium supplemented only with Tf and nutrients without necessity of other serum factor(s) or T3.

Rat pituitary tumor cells in serum-free culture. II. Serum factor and thyroid hormone requirements for estrogen-responsive growth.

The effect of 17 beta-estradiol (E2) on growth of GH4C1 rat pituitary tumor cells was investigated under serum-free conditions and with medium containing charcoal-extracted serum. Serum-free TRM-1 medium was a 1:1 (vol/vol) mixture of F12-DME supplemented with 50 micrograms/ml gentamicin, 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 10 micrograms/ml insulin, 10 micrograms/ml transferrin, 10 ng/ml selenous acid, 10 nM 3,5,3'-triiodothyronine (T3), 50 microM ethanolamine, and 500 micrograms/ml bovine serum albumin. The cells grew continuously in TRM-1 but were E2 responsive only when growth was retarded by reducing the T3 concentration to 10 pM (TRM-MOD). Addition of 1 to 10 nM E2 to TRM-MOD increased growth by 0.3 to 0.9 cell population doublings over controls in 9 d. By using medium supplemented with charcoal-extracted sera, basal growth became 1 to 1.5 cell population doublings in 9 d. Addition of 0.1 pM E2 to medium containing charcoal-extracted serum caused a significant increase in cell number whereas pM-nM concentrations stimulated 200 to 570% increases over controls. The effect of steroid hormone was the same in phenol-red-containing and indicator-free medium. The data presented confirm that the major requirements for demonstration of estrogenic effects in culture were optimum concentrations of thyroid hormones and the presence of yet-to-be-characterized serum factors.