Proc. Nati. Acad. Sci. USA Vol. 75, No. 8, pp. 3786-3790, August 1978
Cell Biology
Estrogen induction of growth factors specific for hormoneresponsive mammary, pituitary, and kidney tumor cells* (rat uterine and kidney activity/hamster liver and kidney activity/target cell specificity/heat stability)
DAVID A. SIRBASKU Department of Biochemistry and Molecular Biology, The University of Texas Medical School at Houston, P. O. Box 20708, Houston, Texas 77025
Communicated by John M. Buchanan, May 2,1978
ABSTRACT The problem of estrogen-promoted tumor cell growth has been studied extensively in an attempt to establish tWe direct mitogenic role of these steroid hormones. We have developed cell lines from three estrogen-responsive tumors or cell populations: the H-301 kidney tumor celks established from a parent estrogen-dependent hamster kidney tumor the GH3/C14 rat pituitary tumor cell line established as a sutline of the orinina GH3 population, and the MTW9/PL mammary cell line developed from a parent estrogen- and prolactin-responsive M1T-W9A carcinogen-induced rat tumor. With all three of these cell lines, we have encountered a paradox: although estrogens are obligatory for tumor formation in vivo, no direct mitogenic effect of estrogens can be shown in culture when assayed by an increase in cell number. We have thus considered the possibility that estrogens may.induce growth factors in vivo that are then responsible for tumor formation by the three cell lines described. Experiments presented in this report show that extracts of rodent uterus, kidney, or liver contain growth activity for these three tumor cell lines, that estrogen treatment causes an increase in tissue content of these activities, and that the estrogen-induced activities are specific for the estrogen-responsive cells. These studies suggest that estrogen-responsive tumor growth in vivo includes the mechanism of estrogenuterus, kidney, or liver - specific growth factors - estrogenresponsive tumor cells. Recent studies of the role of estrogens in induction and promotion of tumor growth have centered on attempts to show a direct mitogenic effect of these steroids on target cells in vitro (1-6). Although considerable progress has been made on the role of estrogens in differentiated gene expression (7, 8), much
remains unclear concerning steroid induction of growth. One of the major questions remaining is whether the growth of the many estrogen-responsive tumors (9-15) can be explained adequately by the direct interaction of estrogens and target cells. An increased growth rate in vitro of a human mammary tumor cell line (MCF-7) has been reported (16) in response to physiological concentrations of estrogens, although other investigators (17) suggest that the response in vitro was complicated by the history of the stock cultures used to initiate growth experiments; another group (18) believes that MCF-7 cells are not growth responsive to estradiol. The problems with estrogen mitogenicity are further emphasized by the reports that estrogens may have a survival or permissive effect in vitro, but little or no positive effect on growth of human mammary tumor explants (4, 6), carcinogen-induced hormone-responsive rat mammary tumor tissue in vitro (3, 19-22), and organ cultures of estrogen-induced kidney tumors (5). The evidence available on the nonestrogen steroid hormones indicates that they act as permissive agents rather than primary mitogens and that continuous growth in the absence of serum or at a limiting serum concentration is promoted by mitogenic mixtures that always include an essential polypeptide factor (23, 24).
To further define the role of estrogens in cell growth, my laboratory has developed permanent cell lines from three-estrogen-responsive rodent tumors: the MTW9/PL estrogen-, thyroid hormone-, and prolactin-responsive rat mammary tumor cells (25); the GH3/C14 estrogen- and thyroid hormone-responsive rat pituitary tumor cells (2, 26, 27); and the H-301 estrogen-responsive hamster kidney line (1). All three established cell lines require estrogens for optimal tumor formation in host animals, but when tested in culture do not respond to estrogens (or prolactin) with increased cell growth rate as measured by increased cell number. These observations presented a paradox. One possible explanation for these apparently contradictory results may be that one of the as yet unrecognized roles of estrogens in vivo is to induce production or secretion of growth factors that enter the general circulation and promote tumor growth. In this report, I have approached estrogen mitogenicity from this new direction by first identifying tissues that contain growth factor activities for the three cell lines, then demonstrating that estrogen treatment influences the tissue levels of these activities, and finally establishing that the estrogen-related growth activities are specific for each type of tumor cell. MATERIALS AND METHODS Cell Lines and Growth Conditions. Conditions have been described for growth and passage of the H-301 hamster kidney tumor cells (1), the GH3/C14 rat pituitary tumor cells (26), and the MTW9/PL rat mammary tumor cells (25). When assays of the mitogenic effects of organ extracts were performed, cells were inoculated first at densities of 3 to 4 X 104 per tissue culture plate (35 X 10 mm) in 3 ml of Dulbecco's modified Eagle's medium containing 10% (vol/vol) fetal calf serum. After 18-24 hr, the medium was removed and replaced with 3.0 ml of serum-free modified Eagle's medium containing the designated concentrations of organ extracts. After 3 days (H-301 cells) or 6 days (MTW9/PL and GH3/C14 cells) at 370 in a humid atmosphere of 95% air/5% CO2, the cell numbers in triplicate test plates were determined by Coulter counter. The BALB/3T3 (A-31) mouse fibroblast cell line, the BHK21 hamster fibroblast line, and the HAK hamster epithelial kidney tumor line were grown in 10% (vol/vol) calf serum. Other lines also obtained from the American Type Culture Collection were the adrenocorticotropin-responsive Y1 mouse adrenal cortex tumor cells (29), the R2C steroid-producing rat Leydig tumor Abbreviations: estradiol, 1,3,5(10)-estratriene-3,17#-diol; MGF, mammary growth factor; PGF, pituitary growth factor; KGF, kidney growth factor. * This report is one of a series describing studies of the hormonal control of cell growth. Other papers of the series are refs. 1, 2, 25, 26, and 27. A preliminary report of these results was presented at the 17th Annual Meeting of the American Society for Cell Biology, San Diego, CA
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
(November, 1977).
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Proc. Natl. Acad. Sci. USA 75 (1978)
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Table 1. Correlation between uterine MGF activity and tumor formation by MTW9/PL mammary tumor cells Estrogen-treated ovariectomized Ovariectomized females females Normal females Uterine mass, 177 ± 34 738 ± 216 658 ± 122 mg ± SD 3.7 1.0 4.4 Relative uterine mass Relative MTW9/PL uterine 3.6 1.0 13.5 growth activity 59.4 1.0 13.3 Total relative activity* Relative mass of MTW9/PL 1.0 40 20 tumors in W/Fu ratst * Total relative activity is the product of the relative mass times the relative activity. t Data taken from ref. 25.
cells (30), the albumin- and complement-secreting MH1C1 rat hepatoma cells (31-34), and the adrenocorticotropin-secreting AtT20 mouse pituitary tumor cells (35). All were grown in Dulbecco's modified Eagle's medium supplemented with 12.5% (vol/vol) horse serum and 2.5% (vol/vol) fetal calf serum. Hormone Treatment of Animals. The rats used were 15- to 18-week-old outbred Sprague-Dawley females obtained from Texas Inbred Mice Company, Houston, TX. Extracts were prepared from the organs and tissues of intact female rats, ovariectomized females, and exogenously estrogen-treated ovariectomized females. Estrogen treatment was by subcutaneous implantation of a single 25-mg pellet consisting of 90% 1,3,5(10)-estratriene-3,17#-diol (estradiol) and 10% cholesterol (wt/wt), which elevated serum estradiol levels 20-30 times above normal levels in female rats (26). Tissues and organs were harvested 30 days after estrogen treatment and/or ovariectomy were begun. The other animal studies were done with 80- to 100-g male Syrian hamsters purchased from ARS SpragueDawley, Madison, WI. Estrogen treatment was as described above. Organs were collected from estradiol-treated males 35 days after pellet implantation, and from untreated males of similar age at the same time. Preparation of Tissue Extracts. Tissues and organs excised from rats or hamsters were rinsed in distilled water to r emove excess blood and homogenized with a Tekmar Tissu.imizer
0% se?rum
- -
5
10 50 100 Uterine protein,,g/ml
FIG. 1. Growth of MTW9/PL cells in response to rat uiterine ter ne extracts. Uterine preparations from normal (M), ovariectomize and estradiol-treated ovariectomized (-) females were added at the designated protein concentrations in serum-free modified E agle's medium, and the cell nu'mbers were determined after 6 days. SD of the means of the cell numbers was ±15% or less.
(Tekmar Company, Cincinnati, OH) in 3 ml of buffer per g of tissue. The buffer used was standard Dulbecco's phosphatebuffered saline (pH 7.2) containing 0.1% disodium EDTA. The homogenates were clarified by centrifugation at 105,000 X g for 90 min at 40 and filter sterilized, and the supernatants were stored at -18°. Protein concentrations in the samples were determined spectrophotometrically by the method of Warburg and Christian (28). Specific Activity of Growth Factors in Organ Extracts. A unit of growth factor activity was defined as the amount that gives one-half the maximal number of cell population doublings at 370 in 3 days for the H-301 kidney cells or 6 days with the MTW9/PL mammary or the GH3/C14 pituitary cells. The specific activity of organ extracts was then expressed as the Ag of protein per ml that was added to the tissue culture medium to give one-half maximal growth. The maximal number of doublings for either MTW9/PL cells or H-301 cells was represented by growth seen in modified Eagle's medium containing 10% (vol/vol) fetal calf serum. Maximal growth for GH3/C14 pituitary cells was shown in modified Eagle's medium containing 12.5% (vol/vol) horse serum and 2.5% (vol/vol) fetal calf serum. RESULTS Rat Tissue Growth Factor Activities for MTW9/PL Mammary Tumor Cells. Extracts of 15 tissues and organs of intact female rats were prepared, and assays showed no significantt mammary growth factor (MGF) activity in any but 3 tissues; the order of relative specific activity in these three was uterus > kidney = brain. Since these are known estrogen target tissues, the effects of exogenous estrogen treatment or estrogen deficiency on the levels of MGF were studied. As shown in Fig. 1, the relative potency of the MGF present in the uterine extracts was estrogen-treated ovariectomized > normal >> ovariectomized females. MGF relative specific activities present in uterine extracts are given in Table 1, demonstrating clearly that the specific activity of uterine-derived MGF is estrogen related in vivo. The uterine mass shows the expected increase or marked decline with estrogen status. Thus, estrogens may have two means of increasing MGF from uterus: elevation of tissue content and an increase in the mass of the producing tissue. The MGF activity from kidney extracts was elevated 2-fold in estrogen-treated ovariectomized rats and normal females as compared to ovariectomized females; there was no significant t Extracts of rat tissues that yielded less than one population doubling
in 6 days when added at a final protein concentration of 500 jg/ml of tissue culture medium were considered to be inactive.
120-
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o-0 x 604 0I
.0
E 501 c
Proc. Natl. Acad. Sci. USA 75 (1978)
Horse-fetal calf serum
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Cell Biology: Sirbasku
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I
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T-t-r--T
10 50 100 Uterine protein,,g/ml
serum
I-
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-r
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FIG. 2. Growth of GH3/C14 cells in response to rat uterine extracts. The uterine preparations from the same three groups of rats shown in Fig. 1 were tested for growth activity as described in that figure legend. Symbols are the same as in Fig. 1.
change in the mass of the rat kidneys. Brain showed MGF activity equal to that of kidney, but there was no significant change caused by estrogen treatment. Extracts of both kidney and brain MGF showed only one-quarter the specific activity of normal uterus. Rat Tissue Growth Activities for GH3/C14 Pituitary Tumor Cells. When extracts from the organs of female rats were tested for pituitary growth factor (PGF) activity with GH3/C14 cells, the tissue distribution of PGF proved very similar to that of MGF activity; the relative specific activities were uterus > kidney = brain. The response pattern was different from that seen with MGF activity, (Fig. 2) since uteri from normal females showed greater PGF activity than those from estrogen-treated ovariectomized females. Nevertheless, uteri from ovariectomized females still showed considerably less growth activity than the other two groups In no case was uterine PGF able to replace completely the serum requirements of GH3/C14 cells. The PGF activity of kidney was also influenced by estrogen, Table 2. Correlation between uterine PGF activity and tumor formation by GH3/C14 pituitary tumor cells Estrogentreated OvariNormal ectomized females females 1.0 3.7
ovariectomized females
4.4 Relative uterine mass Relative GH3/C14 uterine 1.0 0.83 3.6 growth factor activity 3.1 1.0 15.8 Total relative activity* Relative mass of GH3/C14 tumors in W/Fu Ratst 29 1.0 124 * Total relative activity is the product of the relative mass times the relative activity. t Data taken from ref. 26.
5
10 50 Kidney protein,,g/ml
100
200
FIG. 3. Growth of H-301 kidney cells in response to hamster kidney extracts prepared from normal male hamsters (0) and estradiol-treated male hamsters (@). Growth was assayed by addition of protein at the designated concentrations to serum-free modified Eagle's medium. Cell numbers were determinedafter 3 days. SD of the means of the cell numbers was +15% or lw:
although the PGF from female kidneyswas 30-40% as active as that obtained from normal female uteri. Brain PGF activity was not changed by estrogen status. From the relative uterine masses of the three groups of animals (Table 2) it is apparent that uterine-derived PGF activity could be elevated by both an apparent increase in tissue content and an increase in total tissue mass. Hamster Liver and Kidney Growth Activities for H-301 Kidney Tumor Cells. We have shown previously (1) that the H-01 kidney tumor cells will nor fbrm tumors in either normal female or normal male hamsters without treatment with exogenous estrogen.
Organ extracts from 12 tissueswere prepared from normal untreated male hamsters and from male hamsters treated with estradiol pellets. The assays showed substantial kidney growth factor (KGF) activity in only live'and kidney.J KGF activities from both kidney and liver were elevated by estrogen treatment (Fig. 3 and Fig. 4, respectively) and were of approximate equal potency.
Growth Factor Specificity and Iuat Stability. The cell type specificity was tested with extracftVf rat uterus and male hamster liver. For the purposes of the.studies, any tissue extract that yielded only one doubling wt.tiot considered a significant source of growth factor activity. The promotion of a single population doubling could be the result of the pH fluctuation occurring at the time of medium change or the mitogenic effect of tissue proteases in the extracts. As shown in Table 3, extracts of uteri from estrogen-treated ovariectomized rats promoted greater growth of MTW9/PL and GH3/C14 cells than equivalent concentrations of extracts from ovariectomized hosts; these data confirm those shown Fig. 1 and Fig. 2, respectively. In addition, an estrogen-influenced KGF for the hamster H-301 cells was identified in extracts of uteri from estrogen-treated rats that showed elevated KGF activity as compared to ovariectomized controls. When similar uterine extracts were tested for growth activity with the mesenchymal-origin BALB/3T3 cells, no substantial growth was demonstrated. With another mesenchymal-origin t Extracts of hamster tissues that yielded less than two population doublings in 5 days when added at a final protein concentration of 500 ug/ml of tissue culture medium were considered inactive.
Proc. Natl. Acad. Sci. USA 75 (1978)
Cell Biology: Sirbasku 300 280-
10% Fetal calf serum
160t o 140 x t
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E 100
C
o
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5 10 50 Liver protein, jg/ml.
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FIG. 4. Growth of H-301 kidney cells in response to hamster liver extracts prepared from normal male hamsters (0) and estradioltreated male hamsters (0). Growth conditions were identical to those described in the legend of Fig. 3.
cell line, BHK21 hamster fibroblasts, a significant uterine growth activity was found, although this activity was not influenced by estrogen treatment. No additional information is available concerning this BHK21 cell activity, although it may be similar to another mesenchymal cell activity identified in uterine extracts by King et al. (36). In additional studies shown in Table 3, the uterine activity did not promote growth of the epithelial MH1C1 rat hepatoma cells, the R2C rat Leydig tumor cells, or the Y1 mouse adrenal cortex tumor cells. Two other epithelial cell lines, namely, the HAK hamster kidney line and the AtT20 mouse pituitary tumor cells, did respond to the uterine extracts, but again the activity was not estrogen related and was lower than that seen with estrogen-responsive tumor cells. When extracts of estradiol-treated hamster liver were tested with the above cell lines of both epithelial and mesenchymal origin, the hamster liver activity was remarkably specific for
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the H-301 hamster kidney tumor cells. The liver extracts did not promote significant growth of any of the other cell lines tested (Table 3). The heat stability of the rat uterine, hamster kidney, and hamster liver factors was tested; greater than 90% of the MGF and PGF activities in rat uterine extracts could be inactivated by heating for 20 min at 700. The KGF activity from estrogen-treated male hamster liver or kidney was lost completely by heat treatment at 1000 for 15 min. These data, along with the fact that the activities just described are not dialyzable, suggest that the factors are macromolecules. DISCUSSION The problems associated with demonstration of a direct mitogenic effect of estrogens on tumor cells in vitro are apparent from work done in our laboratory (1, 2) as well as other laboratories (3-6, 19-22). These observations have led us to consider alternative mechanisms to the generally accepted model for estrogen interactions with target cells. To date, the most often stated mechanism of estrogen induction of growth has been that estrogens interact with specific cytoplasmic and nuclear receptor proteins, which then become associated with chromatin, causing gene expression and synthesis of specific proteins leading to cell division (7). Nevertheless, studies with many cell types in vitro suggest that growth in response to steroid hormones is limited (3-6, 19-22). Most often, any effective defined growth-promoting medium has been shown to contain at least one essential polypeptide activity (23, 37) or serum. From this starting point, we have attempted to identify possible growth factor activities that are produced in response to estrogen treatment and that are specific for hormone-responsive cells. As can be seen from Table 3, the estrogen-related growth factor activities identified in this report have shown thus far a relative specificity for cells that form estrogen-responsive tumors. The estrogen-inducible activity extracted from rat uterus promotes growth of all three of the estrogen-responsive cell types studied, but not of such estrogen-unresponsive cells as BALB/3T3, R2C, Y1, and MH1C1. Some other cell types, such as BHK21, AtT20, and HAK, show growth responses to uterine extracts, but no significant estrogen effect was seen in these activities. Another important property of the estrogen-related growth factors is the apparent correlation between total relative activity
Table 3. Cell type specificity of growth activities from rat uterus and hamster liver Cell population doublings Cell population doublings in rat uterus extract* in hamster liver extractt Estrogen-treated Cell lines Ovariectomized ovariectomized Normal Estrogen-treated MTW9/PL 2.4 3.4 1.2 1.0 GH3/C14 2.6 3.8 1.5 0.8 H-301 4.2 5.1 2.7 4.0 R2C 0 0 0 0 MH1C1 0.1 0.3 1.0 1.3 Y1 1.2 1.4 0.4 0.3 AtT20 2.4 2.1 0.6 1.0 HAK 2.1 2.2 0.4 0.4 BHK21 2.4 2.6 1.2 0.8 BALB/3T31 1.2 0.6 0.5 0 * Extracts of rat uterus were added at a final protein concentration of 500 ,g/ml. Unless otherwise noted, the cell growth assays were of 6 days' duration with uterine extracts. t Extracts of hamster liver were added at a final protein concentration of 200 ,g/ml. Unless otherwise noted, the cell growth assays were of 3 days' duration with liver extracts. All growth assays with 3T3 cells were of 5 days' duration.
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CeR Biology: Sirbasku
in the uterus and tumor formation by the hormone-responsive tumor cells in host animals. The best examples of this correlation are shown with uterine extracts and MTW9/PL or GH3/C14 tumor formation in W/Fu rats. As seen in Table 1, the total relative MGF activity present in extracts of uteri from normal, ovariectomized, and estrogen-treated ovariectomized females correlates well with the relative masses of the tumors formed by MTW9/PL cells in W/Fu rats. It is recognized, however, that the circulation of the factors must be established before one can conclude that uterine MGF is responsible for mammary tumor growth. Table 2 shows the correlation of uterine PGF and GH3/C14 cell tumor formation in the three groups of rats. Here again, the relationship is apparent between total relative PGF and GH3/C14 tumor formation in W/Fu rats, although the values are not as near as witkuterine MGF and MTW9/PL tumors. These data suggest that other sources of PGF in addition to uterus (i.e., kidney or brain) could be of importance in GH3/C14 tumor formation. The role of the hypothalamus in normal pituitary function is well recognized and suggests that brain PGF may be important even though no significant change in tissue specific activity could be detected with change in estrogen status. On the other hand, the kidney is also a source of estrogen-related PGF and could play a vital role in overall pituitary tumor growth. In conclusion, evidence has been presented that estrogenrelated growth factor activities can be identified in normal estrogen target tissues of rodents and that these activities are specific for hormone-responsive cells. These studies suggest that a possible mechanism for some types of estrogen-responsive tumor cell growth may be estrogens uterus, kidney, or liver specific growth factors -- estrogen-responsive tumor cells. I am grateful to Mrs. Frances E. Leland for her expert technical assistance. This work was supported by Grant BC-255 from the American Cancer Society, Inc. 1. Sirbasku, D. A. & Kirkland, W. L. (1976) Endocrinology 98, 1260-1272. 2. Kirkland, W. L., Sorrentino, J. M. & Sirbasku, D. A. (1976) J. Nati. Cancer Inst. 56, 1159-1164. 3. Hallowes, R. C., Rudland, P. S., Hawkins, R. A., Lewis, D. J., Bennett, D. & Durbin, H. (1977) Cancer Res. 37, 2492-2504. 4. Lewis, D. & Hallowes, R. C. (1976) in Human Tumours in Short Term Culture, ed. Dendy, D. P. (Academic, New York), pp. 219-226. 5. Algard, F. T. (1960) J. Natl. Cancer Inst. 25,557-571. 6. Heuson, J. C., Pasteels, J. L., Legros, N., Heuson-Stiennon, J. & Leclercq, G. (1975) Cancer Res. 35,2039-2048. 7. O'Malley, B. W. & Means, A. R. (1974) Science 183,610-620. 8. Palmiter, R. (1975) Cell 4, 189-197. 9. Hooker, C. W. & Pfeiffer, C. A. (1942) Cancer Res. 2, 759760.
Proc. Nati. Acad. Sci. USA 75 (1978) 10. Allen, E. & Gardner, W. U. (1941) Cancer Res. 1, 359-366. 11. Gardner, W. U., Kirshbaum, A. & Strong, L. C. (1940) Arch. Pathol. 29, 1-7. 12. Kim, U., Furth, J. & Yannopoulos, K. (1964) J. Natl. Cancer Inst. 31,233-259. 13. Redman, L. W., Garland, M. R. & Thomson, M. (1971) Cancer Res. 31,265-269. 14. Kirkman, H. (1959) Natl. Cancer Inst. Monogr. 1, 1-139. 15. Furth, J., Clifton, K. H. & Gadsden, E. B. (1956) Cancer Res. 16, 608-616. 16. Lippman, M. E., Bolan, G. & Huff, K. (1976) Cancer Res. 35, 4595-5601. 17. Butler, W. B., Brooks, S. C. & Goran, N. (1977) J. Cell. Biol. 75, part 2,186a. 18. Zava, D. T., Chamnes, G. C., Harwitz, K. B. & McGuire, W. L. (1977) Science. 196, 663-664. 19. Pasteels, J. L., Heuson, J. C., Heusen-Stiennon, J. & Legros, N. (1976) Cancer Res. 36, 2162-2170. 20. Cohen, L., Tsuang, J. & Chan, P. C. (1974) In Vitro 10, 5162. 21. Welsh, C. W. & Rivera, E. M. (1972) Proc. Soc. Exp. Biol. Med. 139,623-626. 22. Chan, P. C., Tsuang, J., Head, J. & Cohen, L. A. (1976) Proc. Soc. Exp. Biol. Med. 151, 362-365. 23. Gospodarowicz, D., Rudland, P., Lindstrom, J. & Benirschke, K. (1975) in Advances in Metabolic Disorders, eds. Luft, R. & Hall, K. (Academic, New York), pp. 302-5. 24. Cunningham, D. D., Thrash, C. R. & Glynn, R. D. (1974) in Control of Proliferation in Animal Cells, eds. Clarkson, B. & Baserga,.R. (Cold Spring Harbor Laboratory, Cold Spring, NY), pp. 105-113. 25. Sirbasku, D. A. (1978) Cancer Res. 38, 1154-1165. 26. Sorrentino, J. M., Kirkland, W. L. & Sirbasku, D. A. (1976) J. Natl. Cancer Inst. 56, 1149-1154. 27. Sorrentino, J. M., Kirkland, W. L. & Sirbasku, D. A. (1976) J. Natl. Cancer Inst. 56, 1155-1158. 28. Warburg, 0. & Christian; W. (1941) Biochem. Z. 310, 402421. 29. Yasumura, Y., Buonassisi, V. & Sato, G. (1966) Cancer Res. 26, 529-535. 30. Shin, S. I., Yasumura, Y. & Sato, G. H. (1968) Endocrinology 82, 614-616. 31. Rugstad, H. E., Robinson, S. H., Yannoni, C. & Tashjian, A. H.
(1970) Science 170, 553-555. 32. Rommel, F. A., Goldlust, M. B., Bancroft, F. C., Mayer, M. M. & Tashjian, A. H. (1970) J. Immunol. 105,396-403. 33. Richardson, I. U., Tashjian, A. H. & Levine, L. (1969) J. Cell. Biol. 40,236-247. 34. Tashjian, A. H., Bancroft, F. C., Richardson, U. I., Goldlust, M. B., Rommel, F. A. & Ofner, P. (1970) In Vitro 6,32-45. 35. Buonassisi, V., Sato, G. & Cohen, A. I. (1962) Proc. Natl. Acad. Sci. USA 48,1184-1190. 36. King, R. J. B., Kaye, A. M. & Shodell, M. (1977) J. Exp. Cell. Res. 109,1-8. 37. Hayashi, I. & Sato, G. H. (1976) Nature (London) 259, 132134.
Cell Biology
Estrogen induction of growth factors specific for hormoneresponsive mammary, pituitary, and kidney tumor cells* (rat uterine and kidney activity/hamster liver and kidney activity/target cell specificity/heat stability)
DAVID A. SIRBASKU Department of Biochemistry and Molecular Biology, The University of Texas Medical School at Houston, P. O. Box 20708, Houston, Texas 77025
Communicated by John M. Buchanan, May 2,1978
ABSTRACT The problem of estrogen-promoted tumor cell growth has been studied extensively in an attempt to establish tWe direct mitogenic role of these steroid hormones. We have developed cell lines from three estrogen-responsive tumors or cell populations: the H-301 kidney tumor celks established from a parent estrogen-dependent hamster kidney tumor the GH3/C14 rat pituitary tumor cell line established as a sutline of the orinina GH3 population, and the MTW9/PL mammary cell line developed from a parent estrogen- and prolactin-responsive M1T-W9A carcinogen-induced rat tumor. With all three of these cell lines, we have encountered a paradox: although estrogens are obligatory for tumor formation in vivo, no direct mitogenic effect of estrogens can be shown in culture when assayed by an increase in cell number. We have thus considered the possibility that estrogens may.induce growth factors in vivo that are then responsible for tumor formation by the three cell lines described. Experiments presented in this report show that extracts of rodent uterus, kidney, or liver contain growth activity for these three tumor cell lines, that estrogen treatment causes an increase in tissue content of these activities, and that the estrogen-induced activities are specific for the estrogen-responsive cells. These studies suggest that estrogen-responsive tumor growth in vivo includes the mechanism of estrogenuterus, kidney, or liver - specific growth factors - estrogenresponsive tumor cells. Recent studies of the role of estrogens in induction and promotion of tumor growth have centered on attempts to show a direct mitogenic effect of these steroids on target cells in vitro (1-6). Although considerable progress has been made on the role of estrogens in differentiated gene expression (7, 8), much
remains unclear concerning steroid induction of growth. One of the major questions remaining is whether the growth of the many estrogen-responsive tumors (9-15) can be explained adequately by the direct interaction of estrogens and target cells. An increased growth rate in vitro of a human mammary tumor cell line (MCF-7) has been reported (16) in response to physiological concentrations of estrogens, although other investigators (17) suggest that the response in vitro was complicated by the history of the stock cultures used to initiate growth experiments; another group (18) believes that MCF-7 cells are not growth responsive to estradiol. The problems with estrogen mitogenicity are further emphasized by the reports that estrogens may have a survival or permissive effect in vitro, but little or no positive effect on growth of human mammary tumor explants (4, 6), carcinogen-induced hormone-responsive rat mammary tumor tissue in vitro (3, 19-22), and organ cultures of estrogen-induced kidney tumors (5). The evidence available on the nonestrogen steroid hormones indicates that they act as permissive agents rather than primary mitogens and that continuous growth in the absence of serum or at a limiting serum concentration is promoted by mitogenic mixtures that always include an essential polypeptide factor (23, 24).
To further define the role of estrogens in cell growth, my laboratory has developed permanent cell lines from three-estrogen-responsive rodent tumors: the MTW9/PL estrogen-, thyroid hormone-, and prolactin-responsive rat mammary tumor cells (25); the GH3/C14 estrogen- and thyroid hormone-responsive rat pituitary tumor cells (2, 26, 27); and the H-301 estrogen-responsive hamster kidney line (1). All three established cell lines require estrogens for optimal tumor formation in host animals, but when tested in culture do not respond to estrogens (or prolactin) with increased cell growth rate as measured by increased cell number. These observations presented a paradox. One possible explanation for these apparently contradictory results may be that one of the as yet unrecognized roles of estrogens in vivo is to induce production or secretion of growth factors that enter the general circulation and promote tumor growth. In this report, I have approached estrogen mitogenicity from this new direction by first identifying tissues that contain growth factor activities for the three cell lines, then demonstrating that estrogen treatment influences the tissue levels of these activities, and finally establishing that the estrogen-related growth activities are specific for each type of tumor cell. MATERIALS AND METHODS Cell Lines and Growth Conditions. Conditions have been described for growth and passage of the H-301 hamster kidney tumor cells (1), the GH3/C14 rat pituitary tumor cells (26), and the MTW9/PL rat mammary tumor cells (25). When assays of the mitogenic effects of organ extracts were performed, cells were inoculated first at densities of 3 to 4 X 104 per tissue culture plate (35 X 10 mm) in 3 ml of Dulbecco's modified Eagle's medium containing 10% (vol/vol) fetal calf serum. After 18-24 hr, the medium was removed and replaced with 3.0 ml of serum-free modified Eagle's medium containing the designated concentrations of organ extracts. After 3 days (H-301 cells) or 6 days (MTW9/PL and GH3/C14 cells) at 370 in a humid atmosphere of 95% air/5% CO2, the cell numbers in triplicate test plates were determined by Coulter counter. The BALB/3T3 (A-31) mouse fibroblast cell line, the BHK21 hamster fibroblast line, and the HAK hamster epithelial kidney tumor line were grown in 10% (vol/vol) calf serum. Other lines also obtained from the American Type Culture Collection were the adrenocorticotropin-responsive Y1 mouse adrenal cortex tumor cells (29), the R2C steroid-producing rat Leydig tumor Abbreviations: estradiol, 1,3,5(10)-estratriene-3,17#-diol; MGF, mammary growth factor; PGF, pituitary growth factor; KGF, kidney growth factor. * This report is one of a series describing studies of the hormonal control of cell growth. Other papers of the series are refs. 1, 2, 25, 26, and 27. A preliminary report of these results was presented at the 17th Annual Meeting of the American Society for Cell Biology, San Diego, CA
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(November, 1977).
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Table 1. Correlation between uterine MGF activity and tumor formation by MTW9/PL mammary tumor cells Estrogen-treated ovariectomized Ovariectomized females females Normal females Uterine mass, 177 ± 34 738 ± 216 658 ± 122 mg ± SD 3.7 1.0 4.4 Relative uterine mass Relative MTW9/PL uterine 3.6 1.0 13.5 growth activity 59.4 1.0 13.3 Total relative activity* Relative mass of MTW9/PL 1.0 40 20 tumors in W/Fu ratst * Total relative activity is the product of the relative mass times the relative activity. t Data taken from ref. 25.
cells (30), the albumin- and complement-secreting MH1C1 rat hepatoma cells (31-34), and the adrenocorticotropin-secreting AtT20 mouse pituitary tumor cells (35). All were grown in Dulbecco's modified Eagle's medium supplemented with 12.5% (vol/vol) horse serum and 2.5% (vol/vol) fetal calf serum. Hormone Treatment of Animals. The rats used were 15- to 18-week-old outbred Sprague-Dawley females obtained from Texas Inbred Mice Company, Houston, TX. Extracts were prepared from the organs and tissues of intact female rats, ovariectomized females, and exogenously estrogen-treated ovariectomized females. Estrogen treatment was by subcutaneous implantation of a single 25-mg pellet consisting of 90% 1,3,5(10)-estratriene-3,17#-diol (estradiol) and 10% cholesterol (wt/wt), which elevated serum estradiol levels 20-30 times above normal levels in female rats (26). Tissues and organs were harvested 30 days after estrogen treatment and/or ovariectomy were begun. The other animal studies were done with 80- to 100-g male Syrian hamsters purchased from ARS SpragueDawley, Madison, WI. Estrogen treatment was as described above. Organs were collected from estradiol-treated males 35 days after pellet implantation, and from untreated males of similar age at the same time. Preparation of Tissue Extracts. Tissues and organs excised from rats or hamsters were rinsed in distilled water to r emove excess blood and homogenized with a Tekmar Tissu.imizer
0% se?rum
- -
5
10 50 100 Uterine protein,,g/ml
FIG. 1. Growth of MTW9/PL cells in response to rat uiterine ter ne extracts. Uterine preparations from normal (M), ovariectomize and estradiol-treated ovariectomized (-) females were added at the designated protein concentrations in serum-free modified E agle's medium, and the cell nu'mbers were determined after 6 days. SD of the means of the cell numbers was ±15% or less.
(Tekmar Company, Cincinnati, OH) in 3 ml of buffer per g of tissue. The buffer used was standard Dulbecco's phosphatebuffered saline (pH 7.2) containing 0.1% disodium EDTA. The homogenates were clarified by centrifugation at 105,000 X g for 90 min at 40 and filter sterilized, and the supernatants were stored at -18°. Protein concentrations in the samples were determined spectrophotometrically by the method of Warburg and Christian (28). Specific Activity of Growth Factors in Organ Extracts. A unit of growth factor activity was defined as the amount that gives one-half the maximal number of cell population doublings at 370 in 3 days for the H-301 kidney cells or 6 days with the MTW9/PL mammary or the GH3/C14 pituitary cells. The specific activity of organ extracts was then expressed as the Ag of protein per ml that was added to the tissue culture medium to give one-half maximal growth. The maximal number of doublings for either MTW9/PL cells or H-301 cells was represented by growth seen in modified Eagle's medium containing 10% (vol/vol) fetal calf serum. Maximal growth for GH3/C14 pituitary cells was shown in modified Eagle's medium containing 12.5% (vol/vol) horse serum and 2.5% (vol/vol) fetal calf serum. RESULTS Rat Tissue Growth Factor Activities for MTW9/PL Mammary Tumor Cells. Extracts of 15 tissues and organs of intact female rats were prepared, and assays showed no significantt mammary growth factor (MGF) activity in any but 3 tissues; the order of relative specific activity in these three was uterus > kidney = brain. Since these are known estrogen target tissues, the effects of exogenous estrogen treatment or estrogen deficiency on the levels of MGF were studied. As shown in Fig. 1, the relative potency of the MGF present in the uterine extracts was estrogen-treated ovariectomized > normal >> ovariectomized females. MGF relative specific activities present in uterine extracts are given in Table 1, demonstrating clearly that the specific activity of uterine-derived MGF is estrogen related in vivo. The uterine mass shows the expected increase or marked decline with estrogen status. Thus, estrogens may have two means of increasing MGF from uterus: elevation of tissue content and an increase in the mass of the producing tissue. The MGF activity from kidney extracts was elevated 2-fold in estrogen-treated ovariectomized rats and normal females as compared to ovariectomized females; there was no significant t Extracts of rat tissues that yielded less than one population doubling
in 6 days when added at a final protein concentration of 500 jg/ml of tissue culture medium were considered to be inactive.
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Proc. Natl. Acad. Sci. USA 75 (1978)
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-r
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FIG. 2. Growth of GH3/C14 cells in response to rat uterine extracts. The uterine preparations from the same three groups of rats shown in Fig. 1 were tested for growth activity as described in that figure legend. Symbols are the same as in Fig. 1.
change in the mass of the rat kidneys. Brain showed MGF activity equal to that of kidney, but there was no significant change caused by estrogen treatment. Extracts of both kidney and brain MGF showed only one-quarter the specific activity of normal uterus. Rat Tissue Growth Activities for GH3/C14 Pituitary Tumor Cells. When extracts from the organs of female rats were tested for pituitary growth factor (PGF) activity with GH3/C14 cells, the tissue distribution of PGF proved very similar to that of MGF activity; the relative specific activities were uterus > kidney = brain. The response pattern was different from that seen with MGF activity, (Fig. 2) since uteri from normal females showed greater PGF activity than those from estrogen-treated ovariectomized females. Nevertheless, uteri from ovariectomized females still showed considerably less growth activity than the other two groups In no case was uterine PGF able to replace completely the serum requirements of GH3/C14 cells. The PGF activity of kidney was also influenced by estrogen, Table 2. Correlation between uterine PGF activity and tumor formation by GH3/C14 pituitary tumor cells Estrogentreated OvariNormal ectomized females females 1.0 3.7
ovariectomized females
4.4 Relative uterine mass Relative GH3/C14 uterine 1.0 0.83 3.6 growth factor activity 3.1 1.0 15.8 Total relative activity* Relative mass of GH3/C14 tumors in W/Fu Ratst 29 1.0 124 * Total relative activity is the product of the relative mass times the relative activity. t Data taken from ref. 26.
5
10 50 Kidney protein,,g/ml
100
200
FIG. 3. Growth of H-301 kidney cells in response to hamster kidney extracts prepared from normal male hamsters (0) and estradiol-treated male hamsters (@). Growth was assayed by addition of protein at the designated concentrations to serum-free modified Eagle's medium. Cell numbers were determinedafter 3 days. SD of the means of the cell numbers was +15% or lw:
although the PGF from female kidneyswas 30-40% as active as that obtained from normal female uteri. Brain PGF activity was not changed by estrogen status. From the relative uterine masses of the three groups of animals (Table 2) it is apparent that uterine-derived PGF activity could be elevated by both an apparent increase in tissue content and an increase in total tissue mass. Hamster Liver and Kidney Growth Activities for H-301 Kidney Tumor Cells. We have shown previously (1) that the H-01 kidney tumor cells will nor fbrm tumors in either normal female or normal male hamsters without treatment with exogenous estrogen.
Organ extracts from 12 tissueswere prepared from normal untreated male hamsters and from male hamsters treated with estradiol pellets. The assays showed substantial kidney growth factor (KGF) activity in only live'and kidney.J KGF activities from both kidney and liver were elevated by estrogen treatment (Fig. 3 and Fig. 4, respectively) and were of approximate equal potency.
Growth Factor Specificity and Iuat Stability. The cell type specificity was tested with extracftVf rat uterus and male hamster liver. For the purposes of the.studies, any tissue extract that yielded only one doubling wt.tiot considered a significant source of growth factor activity. The promotion of a single population doubling could be the result of the pH fluctuation occurring at the time of medium change or the mitogenic effect of tissue proteases in the extracts. As shown in Table 3, extracts of uteri from estrogen-treated ovariectomized rats promoted greater growth of MTW9/PL and GH3/C14 cells than equivalent concentrations of extracts from ovariectomized hosts; these data confirm those shown Fig. 1 and Fig. 2, respectively. In addition, an estrogen-influenced KGF for the hamster H-301 cells was identified in extracts of uteri from estrogen-treated rats that showed elevated KGF activity as compared to ovariectomized controls. When similar uterine extracts were tested for growth activity with the mesenchymal-origin BALB/3T3 cells, no substantial growth was demonstrated. With another mesenchymal-origin t Extracts of hamster tissues that yielded less than two population doublings in 5 days when added at a final protein concentration of 500 ug/ml of tissue culture medium were considered inactive.
Proc. Natl. Acad. Sci. USA 75 (1978)
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160t o 140 x t
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C
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FIG. 4. Growth of H-301 kidney cells in response to hamster liver extracts prepared from normal male hamsters (0) and estradioltreated male hamsters (0). Growth conditions were identical to those described in the legend of Fig. 3.
cell line, BHK21 hamster fibroblasts, a significant uterine growth activity was found, although this activity was not influenced by estrogen treatment. No additional information is available concerning this BHK21 cell activity, although it may be similar to another mesenchymal cell activity identified in uterine extracts by King et al. (36). In additional studies shown in Table 3, the uterine activity did not promote growth of the epithelial MH1C1 rat hepatoma cells, the R2C rat Leydig tumor cells, or the Y1 mouse adrenal cortex tumor cells. Two other epithelial cell lines, namely, the HAK hamster kidney line and the AtT20 mouse pituitary tumor cells, did respond to the uterine extracts, but again the activity was not estrogen related and was lower than that seen with estrogen-responsive tumor cells. When extracts of estradiol-treated hamster liver were tested with the above cell lines of both epithelial and mesenchymal origin, the hamster liver activity was remarkably specific for
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the H-301 hamster kidney tumor cells. The liver extracts did not promote significant growth of any of the other cell lines tested (Table 3). The heat stability of the rat uterine, hamster kidney, and hamster liver factors was tested; greater than 90% of the MGF and PGF activities in rat uterine extracts could be inactivated by heating for 20 min at 700. The KGF activity from estrogen-treated male hamster liver or kidney was lost completely by heat treatment at 1000 for 15 min. These data, along with the fact that the activities just described are not dialyzable, suggest that the factors are macromolecules. DISCUSSION The problems associated with demonstration of a direct mitogenic effect of estrogens on tumor cells in vitro are apparent from work done in our laboratory (1, 2) as well as other laboratories (3-6, 19-22). These observations have led us to consider alternative mechanisms to the generally accepted model for estrogen interactions with target cells. To date, the most often stated mechanism of estrogen induction of growth has been that estrogens interact with specific cytoplasmic and nuclear receptor proteins, which then become associated with chromatin, causing gene expression and synthesis of specific proteins leading to cell division (7). Nevertheless, studies with many cell types in vitro suggest that growth in response to steroid hormones is limited (3-6, 19-22). Most often, any effective defined growth-promoting medium has been shown to contain at least one essential polypeptide activity (23, 37) or serum. From this starting point, we have attempted to identify possible growth factor activities that are produced in response to estrogen treatment and that are specific for hormone-responsive cells. As can be seen from Table 3, the estrogen-related growth factor activities identified in this report have shown thus far a relative specificity for cells that form estrogen-responsive tumors. The estrogen-inducible activity extracted from rat uterus promotes growth of all three of the estrogen-responsive cell types studied, but not of such estrogen-unresponsive cells as BALB/3T3, R2C, Y1, and MH1C1. Some other cell types, such as BHK21, AtT20, and HAK, show growth responses to uterine extracts, but no significant estrogen effect was seen in these activities. Another important property of the estrogen-related growth factors is the apparent correlation between total relative activity
Table 3. Cell type specificity of growth activities from rat uterus and hamster liver Cell population doublings Cell population doublings in rat uterus extract* in hamster liver extractt Estrogen-treated Cell lines Ovariectomized ovariectomized Normal Estrogen-treated MTW9/PL 2.4 3.4 1.2 1.0 GH3/C14 2.6 3.8 1.5 0.8 H-301 4.2 5.1 2.7 4.0 R2C 0 0 0 0 MH1C1 0.1 0.3 1.0 1.3 Y1 1.2 1.4 0.4 0.3 AtT20 2.4 2.1 0.6 1.0 HAK 2.1 2.2 0.4 0.4 BHK21 2.4 2.6 1.2 0.8 BALB/3T31 1.2 0.6 0.5 0 * Extracts of rat uterus were added at a final protein concentration of 500 ,g/ml. Unless otherwise noted, the cell growth assays were of 6 days' duration with uterine extracts. t Extracts of hamster liver were added at a final protein concentration of 200 ,g/ml. Unless otherwise noted, the cell growth assays were of 3 days' duration with liver extracts. All growth assays with 3T3 cells were of 5 days' duration.
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in the uterus and tumor formation by the hormone-responsive tumor cells in host animals. The best examples of this correlation are shown with uterine extracts and MTW9/PL or GH3/C14 tumor formation in W/Fu rats. As seen in Table 1, the total relative MGF activity present in extracts of uteri from normal, ovariectomized, and estrogen-treated ovariectomized females correlates well with the relative masses of the tumors formed by MTW9/PL cells in W/Fu rats. It is recognized, however, that the circulation of the factors must be established before one can conclude that uterine MGF is responsible for mammary tumor growth. Table 2 shows the correlation of uterine PGF and GH3/C14 cell tumor formation in the three groups of rats. Here again, the relationship is apparent between total relative PGF and GH3/C14 tumor formation in W/Fu rats, although the values are not as near as witkuterine MGF and MTW9/PL tumors. These data suggest that other sources of PGF in addition to uterus (i.e., kidney or brain) could be of importance in GH3/C14 tumor formation. The role of the hypothalamus in normal pituitary function is well recognized and suggests that brain PGF may be important even though no significant change in tissue specific activity could be detected with change in estrogen status. On the other hand, the kidney is also a source of estrogen-related PGF and could play a vital role in overall pituitary tumor growth. In conclusion, evidence has been presented that estrogenrelated growth factor activities can be identified in normal estrogen target tissues of rodents and that these activities are specific for hormone-responsive cells. These studies suggest that a possible mechanism for some types of estrogen-responsive tumor cell growth may be estrogens uterus, kidney, or liver specific growth factors -- estrogen-responsive tumor cells. I am grateful to Mrs. Frances E. Leland for her expert technical assistance. This work was supported by Grant BC-255 from the American Cancer Society, Inc. 1. Sirbasku, D. A. & Kirkland, W. L. (1976) Endocrinology 98, 1260-1272. 2. Kirkland, W. L., Sorrentino, J. M. & Sirbasku, D. A. (1976) J. Nati. Cancer Inst. 56, 1159-1164. 3. Hallowes, R. C., Rudland, P. S., Hawkins, R. A., Lewis, D. J., Bennett, D. & Durbin, H. (1977) Cancer Res. 37, 2492-2504. 4. Lewis, D. & Hallowes, R. C. (1976) in Human Tumours in Short Term Culture, ed. Dendy, D. P. (Academic, New York), pp. 219-226. 5. Algard, F. T. (1960) J. Natl. Cancer Inst. 25,557-571. 6. Heuson, J. C., Pasteels, J. L., Legros, N., Heuson-Stiennon, J. & Leclercq, G. (1975) Cancer Res. 35,2039-2048. 7. O'Malley, B. W. & Means, A. R. (1974) Science 183,610-620. 8. Palmiter, R. (1975) Cell 4, 189-197. 9. Hooker, C. W. & Pfeiffer, C. A. (1942) Cancer Res. 2, 759760.
Proc. Nati. Acad. Sci. USA 75 (1978) 10. Allen, E. & Gardner, W. U. (1941) Cancer Res. 1, 359-366. 11. Gardner, W. U., Kirshbaum, A. & Strong, L. C. (1940) Arch. Pathol. 29, 1-7. 12. Kim, U., Furth, J. & Yannopoulos, K. (1964) J. Natl. Cancer Inst. 31,233-259. 13. Redman, L. W., Garland, M. R. & Thomson, M. (1971) Cancer Res. 31,265-269. 14. Kirkman, H. (1959) Natl. Cancer Inst. Monogr. 1, 1-139. 15. Furth, J., Clifton, K. H. & Gadsden, E. B. (1956) Cancer Res. 16, 608-616. 16. Lippman, M. E., Bolan, G. & Huff, K. (1976) Cancer Res. 35, 4595-5601. 17. Butler, W. B., Brooks, S. C. & Goran, N. (1977) J. Cell. Biol. 75, part 2,186a. 18. Zava, D. T., Chamnes, G. C., Harwitz, K. B. & McGuire, W. L. (1977) Science. 196, 663-664. 19. Pasteels, J. L., Heuson, J. C., Heusen-Stiennon, J. & Legros, N. (1976) Cancer Res. 36, 2162-2170. 20. Cohen, L., Tsuang, J. & Chan, P. C. (1974) In Vitro 10, 5162. 21. Welsh, C. W. & Rivera, E. M. (1972) Proc. Soc. Exp. Biol. Med. 139,623-626. 22. Chan, P. C., Tsuang, J., Head, J. & Cohen, L. A. (1976) Proc. Soc. Exp. Biol. Med. 151, 362-365. 23. Gospodarowicz, D., Rudland, P., Lindstrom, J. & Benirschke, K. (1975) in Advances in Metabolic Disorders, eds. Luft, R. & Hall, K. (Academic, New York), pp. 302-5. 24. Cunningham, D. D., Thrash, C. R. & Glynn, R. D. (1974) in Control of Proliferation in Animal Cells, eds. Clarkson, B. & Baserga,.R. (Cold Spring Harbor Laboratory, Cold Spring, NY), pp. 105-113. 25. Sirbasku, D. A. (1978) Cancer Res. 38, 1154-1165. 26. Sorrentino, J. M., Kirkland, W. L. & Sirbasku, D. A. (1976) J. Natl. Cancer Inst. 56, 1149-1154. 27. Sorrentino, J. M., Kirkland, W. L. & Sirbasku, D. A. (1976) J. Natl. Cancer Inst. 56, 1155-1158. 28. Warburg, 0. & Christian; W. (1941) Biochem. Z. 310, 402421. 29. Yasumura, Y., Buonassisi, V. & Sato, G. (1966) Cancer Res. 26, 529-535. 30. Shin, S. I., Yasumura, Y. & Sato, G. H. (1968) Endocrinology 82, 614-616. 31. Rugstad, H. E., Robinson, S. H., Yannoni, C. & Tashjian, A. H.
(1970) Science 170, 553-555. 32. Rommel, F. A., Goldlust, M. B., Bancroft, F. C., Mayer, M. M. & Tashjian, A. H. (1970) J. Immunol. 105,396-403. 33. Richardson, I. U., Tashjian, A. H. & Levine, L. (1969) J. Cell. Biol. 40,236-247. 34. Tashjian, A. H., Bancroft, F. C., Richardson, U. I., Goldlust, M. B., Rommel, F. A. & Ofner, P. (1970) In Vitro 6,32-45. 35. Buonassisi, V., Sato, G. & Cohen, A. I. (1962) Proc. Natl. Acad. Sci. USA 48,1184-1190. 36. King, R. J. B., Kaye, A. M. & Shodell, M. (1977) J. Exp. Cell. Res. 109,1-8. 37. Hayashi, I. & Sato, G. H. (1976) Nature (London) 259, 132134.
