Changes of Endocrine Properties of a Transplantable, Multihormonal, Pituitary Tumor (MtT-F4) After Hypophysectomy of Host Rats M. LIS, M. C. GUERINOT AND M. CHRETIEN Clinical Research Institute of Montreal and Department of Medicine, Universite de Montreal, Montreal, Canada
Y
ABSTRACT. The transplantable rat pituitary tumor MtT-F4 failed to grow in rats hypophysectomized at the time of transplantation, but did grow in thyroxine-treated hypophysectomized rats. In the latter rats, the tumor did not stimulate the adrenals to the same extent as in control rats. When the tumor cells were isolated and incubated in vitro, those from hypophysectomized thyroxine-treated rats released much less ACTH into the incubation medium than the tumor cells from control rats. They
also released significantly less growth hormone than tumor cells from intact, intact thyroxine-treated, and thyroidectomized thyroxine-treated rats. Prolactin release by the isolated tumor cells in vitro was the same in all groups studied. The results suggest that the hypophysectomy and thyroxine treatment of the host rat might selectively influence the production of hormones by the MtT-F4 transplantable rat pituitary tumor. (Endocrinology 96: 739, 1975)
'RANSPLANT/ABLE pituitary tumors hormone (5) we determined the production developed by Furth (1) offer a means of these hormones by tumors grown in of studying the secretion and biosynthesis hypophysectomized and thyroxine-treated of pituitary hormones. The secretion of rats in order to compare them with the pituitary hormones by tumor cells differs, endocrine properties of tumors grown in however, from that of normal pituitary intact rats. since the tumor lacks the hypothalamic control that is essential for normal pituitary Materials and Methods function (2). On the other hand, transRats bearing MtT-F4 tumors were obtained planted tumors and their endocrine ac- from Dr. A. E. Bogden, Mason Research Institivities are not entirely free from external tute, Worcester, Massachusetts, who performed influence. For example, estradiol stimu- the first passage from frozen tumors into 100 g lates prolactin production by tumors while female Fischer 344 rats. Before these tumors inhibiting their effect on the enlargement were used for our experiments they were transof adrenals (3). The AtT-20 type of mouse planted again at least 6 more times. For transthe tumor was cut into pieces about pituitary tumor responds to glucocorticoid plantation, 3 1-2 mm across and suspended and washed in treatment by lowering ACTH production (4). medium 199 (Grand Island Biological ComWe have found that the MtT-F4 type of pany). The tumor fragments were then introtransplantable pituitary tumor fails to duced subcutaneously under light ether anesgrow in rats, hypophysectomized at the thesia through a small incision in the lateral time of transplantation, at least during the abdominal region. In experimental groups to be first 30 days after transplantation; tumor compared, all the tumors originated from the growth is restored by injections of transplanted fragments of one tumor. HypoL-thyroxine. As the MtT-F4 tumor is known physectomy (by the transauricular approach), to produce ACTH, prolactin, and growth ovariectomy, thyroidectomy and adrenaly P
Received July 3, 1974. Work supported by grant 4881 of the Medical Research Council of Canada and the National Cancer Institute of Canada.
ectomy were performed under ether anesthesia. Controls were sham-operated. Completeness of hypophysectomy was verified at autopsy. L-thyroxine (Sigma) was injected subcutaneously in a dose of 10 /xg per rat per day. 739
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Endo • 1975 Vol 96 • No 3
LIS, GUERINOT AND CHRETIEN
740
The tumor cells were isolated by collagenase digestion (M. Lis and M. Chretien, manuscript in preparation). Animals were killed by decapitation and tumors were immediately dissected into pieces about 1-2 mm3 across, washed in saline, and incubated in 10 ml of Ringer-Krebs bicarbonate-glucose (1 mg glucose per ml) containing 30 mg of collagenase (Worthington). After 15 min of incubation at 37 C with 5% CO 2 -95% O2 as a gas phase, 10 ml of RingerKrebs bicarbonate-glucose containing 10 mg of bovine serum albumin (Fraction V, Pentex), 40 fxg of DNase (Sigma), and 20 /u-g of RNase (Sigma) was added. After a further 15 minutes' incubation the tissue fragments were washed and the cells mechanically dispersed in ice-cold Ringer-Krebs bicarbonate-glucose. The cells were washed twice with 50 ml of the same buffer and once with incubation medium. Incubation medium contained bovine serum albumin (2 mg per ml) and lima bean trypsin inhibitor (Worthington) (0.2 mg per ml) in a mixture of Ringer-Krebs bicarbonate-glucose and synthetic medium 199 in the proportions 4:1. In all experiments the cells were isolated and subsequently incubated in Teflon as was recommended by Sayers et al. (15) for isolated adrenal cells. The integrity of cell membranes of incubated cells was established by the exclusion of trypan blue dye. The concentration of cells was determined by means of a hemocytometer and by the measurement of DNA using the ethydium bromide fluorescence method (6). ACTH was determined in the incubation medium by means of a bioassay using isolated rat adrenal cells in vitro essentially as described by Free (7). As ACTH
standard we used synthetic 1-24 peptide (Cosyntropin, Organon, lot #26153). Prolactin and growth hormone were determined in the incubation medium by a radioimmunoassay using rat prolactin and rat growth hormone radioimmunoassay kits distributed by NIAMD. The prolactin and growth hormone were assayed essentially as described in the recommendations accompanying both radioimmunoassay kits. As the iodinated antigens NIAMD-rat prolactin-I-1 was used for prolactin radioimmunoassay, and NIAMD-rat GH-I-2 for growth hormone radioimmunoassay. As reference preparations we used NIAMD-rat prolactin-RP-1 and NIAMD-rat GH-RP-1, respectively. In the prolactin assay we used a double antibody method. In the growth hormone assay we precipitated the antigen-antibody complex with polyethylene-glycol (8). Corticosterone in rat plasma was determined by a protein-binding method essentially as described by Murphy (9).
Results
Table 1 shows that hypophysectomy and thyroidectomy completely prevented the growth of transplanted MtT-F4 tumors, ovariectomy has no effect and adrenalectomy stimulated growth. The weight of adrenals was increased in tumor-bearing rats with corresponding higher corticosterone concentrations in plasma. Thymus weight was decreased by tumor in the presence of adrenals. In adrenalectomized animals, however, the tumor seemed to increase thymus weight.
TABLE 1. Observations on rats with transplanted MtT-F4 tumors Weight Tumor (g)
Control 1.66 ±0.30
Hypophysectomized
Thyroidectomized
Ovariectomized
Adrenalectomized
0
0
2.55 ±0.41
9.4* ±3.4
Adrenals (mg)
171.0 ± 17.7
17.4** ±1.6
Thymus (mg)
44.6 ±6.8
200.0** ±18.7
Corticosterone /i.g/100 ml plasma
45.0 ±4.9
4.15** ±1.24
27.5** ±2.8 95.0* ±14.9 14.4** ±4.6
174 ±22
0
23.5 ±4.7
398** ±29
59.7* ±2.6
2.7** ±2.4
The rats were killed 30 days after tumor transplantation. All data are the means ± standard error. There were 12 rats in each group. Significantly different results, as compared to control group, are indicated by *(95%) and **(99%).
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MtT-F4 TUMOR AFTER HYPOPHYSECTOMY
741
TABLE 2. Effect of L-thyroxine injection on rats with transplanted MtT)F4 tumors Weight
Hypophysectomized
Control
Tumor (g)
162 ±21
Thymus (mg)
81.4 ± 14.3
Thyroidectomized + T4
1.20 ±0.37
1.52 ±0.19
0
1.77 ±0.48
Adrenals (mg)
Hypophysectomized + T4
35.5** ±5.4
12.8** ± 1.0 184** ± 16
236** ±20
119 ±22 92.1 ±17.5
Conditions and statistics as in Table 1 (but separate experiment). T4 = L-thyroxine, 10 /xg/day SC for 30 days.
Daily administration of 10 fig L-thyroxine (T4) permitted tumor growth in hypophysectomized animals (Table 2), but the adrenals were not stimulated and the thymus did not regress as in the control tumorbearing rats. In the thyroidectomized T4treated rats, tumors grew well, the adrenals 0.3r
were stimulated, and the thymus regressed. This difference suggests a decreased ACTH production by tumor tissue in the hypophysectomized groups. This was confirmed by incubation of the tumor cells isolated from these rats and measurement of the ACTH released (Fig. 1). We also found (Fig. 2) that isolated tumor cells from thyroxine-treated, hypophysectomized rats released substantially less growth hormone than did corresponding cells from the other three groups, and this accords well with the substantially lower plasma concentration of growth hormone in these animals (Table 3). The amounts of
* - *
TUMOR CELLS M I T - F j i H p i ; T«
O—6 TUMOR CELLS MIT-F4; Ttii; T/| •—•
TUMOR CELLS MIT-F4; T4
O - O TUMOR CELLS MIT-F*
TUMOR CELLS M»T-F 4 :Hp«:T4
2
4
TIME OF INCUBATION (hours)
FIG. 1. ACTH release by isolated tumor cells. The isolated cells from 4 tumors from different rats in each group were incubated in the medium described under Materials and Methods and incubation medium was collected at the intervals indicated. Aliquots of this medium were then incubated with isolated rat adrenal cells, and corticosterone production was related (see ref. 7) to ACTH standards (synthetic 1-24). All values are the means of two measurements. The experiment was repeated with essentially the same results. Hpx; T4) hypophysectomized host rats treated with L-thyroxine.
TIME OF INCUBATION (hours)
FIG. 2. Growth hormone released by isolated tumor cells. In each incubation the isolated cells from 2 tumors were used. Growth hormone was measured in the incubation medium by radioimmunoassay. All measurements are duplicates and the results are the means of 4 different incubations ± standard error. Hpx, hypophysectomy; T4, L-thyroxine treatment; Thx, thyroidectomy.
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Endo • 1975 Vol 96 • No 3
LIS, GUERINOT AND CHRETIEN
742
prolactin released by the tumor cells from
inhibited tumor growth (rat MtT-F4) and the inhibition was prevented by thyroxine. In our experiments there was no palpable MtT-F4 growth for 30 days after transplantation in thyroidectomized or hypophysectomized rats, while all other groups produced tumors weighing more than 1 g. The difference may lie in the fact that we performed the operations and started thyroxine treatment at the time of transplantation. Mizuno et al. (3) started the thyroxine or thiouracil treatment as late as 4-5 weeks after transplantation. An in vitro stimulating action of thyroxine and L-triiodothyronine on glucose utilization by
all rats were the same (Fig. 3) as were the
cultured pituitary tumor (GH, clone) has
TABLE 3. Plasma growth hormone concentrations of tumor-bearing rats Group Tumor Tumor + T4 Tumor + Hpx + T4 Tumor + Thx + T4
GH (ng/ml plasma) 205 ± 255 ± 144 ± 208 ±
15 11 11* 21
(7) (8) (8) (7)
* Significantly different (P < 0.05). Blood samples were collected under light ether narcosis from jugular vein 26 days after tumor transplantation. GH was measured by radioimmunoassay. The plasma concentrations were correlated to lg of tumor weight. Means ± SE (number of observations in parenthesis).
plasma prolactin concentrations (not shown).
recently been reported (12). The thyroid hormones can influence normal pituitaries: for example, thyroxine stimulates prolactin Discussion release by rat anterior pituitaries in vitro (13). Moreover, thyroidectomy caused a Transplantable mammotropic pituitary gradual decrease in the population of tumors were developed in rats by Furth somatotrophic cells (14). The effect reand coworkers (10). The MtT-F4 line of ported in our experiments does not seem to transplantable pituitary tumor was dea simple effect of thyroxine treatment. be veloped in rats by the implantation of The treatment of intact tumor-bearing rats stilbestrol pellets. At first, these tumors grew only in estrogen-treated hosts, but with thyroxine, or the treatment of subpassaging produced an autonomous var- thyroidectomized tumor-bearing rats with iant which grows in intact Fischer rats (10). thyroxine did not cause any detectable The MtT-F4 tumors were reported to changes when compared with controls (Tasecrete ACTH, prolactin, and growth hor- bles 2 and 3, Fig. 2). Thyroxine treatment mone and to increase plasma concentrations of these substances 6000 times, 50 A — A TUMOR CELLS MtT-F« , Hpi, T4 times, and 30 times, respectively (5). AlA — A TUMOR CELLS MIT-F4 ;TI>« j T • — • TUMOR CELLS MlT-F ;T though the tumors grew autonomously, O — O TUMOR CELLS MlT-F several workers noted that changing the hormonal balance of host rats or of the incubation medium could influence the growth and endocrine properties of these tumors. For example, estradiol, cortisol, and thyroxine treatment of rat bearing MtT-F4 tumors raised plasma prolactin concentrations (3). There are, however, some discrepancies between our results and those of others. For example Mizuno et TIME OF INCUBATION (hours) al. (3) reported no influence of thyroxine or thiouracil on MtT-F4 tumor growth, while FIG. 3. Prolactin released by isolated tumor cells. was measured by radioimmunoassay in the Milkovic et al. (11) found that thyroidec- Prolactin same incubation medium samples as were used for tomy or thiouracil treatment significantly Fig. 2. 4
4
4
4
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MtT-F4 TUMOR AFTER HYPOPHYSECTOMY was sufficient to restore tumor growth in hypophysectomized animals, but apparently failed to produce the usual endocrine profile of tumors grown in intact hosts. The nature of the factor(s) required for the maintenance of the usual endocrine pattern of MtT-F4 tumors remains to be elucidated. Acknowledgments The authors thank Mrs. F. Rousseau for her excellent technical assistance. The secretarial help of Mrs. D. Marcil is also appreciated. The authors are indebted to NIAMD for providing the radioimmunoassay kits for rat prolactin and rat growth hormone, to Dr. A. E. Bogden, Mason Research Institute for MtT-F4 tumor, and to Organon Inc. for the gift of Cosyntropin peptide.
References 1. Furth, J., Recent Progr Horm Res 11: 221, 1955. 2. Ueda, G., S. Takizawa, P. Moy, F. Marolla, and J. Furth, Cancer Res 28: 1963, 1968.
743
3. Mizuno, H., P. K. Talwalker, and J. Meites, Cancer Res 24: 1433, 1964. 4. Watanabe, H., W. E. Nicholson, and D. N. Orth, Endocrinology 93: 411, 1973. 5. Bates, R. W., S. Milkovic, and M. M. Garrison, Endocrinology 71: 943, 1962. 6. Prasad, A. S., E. DuMouchelle, D. Koniuch, and D. OberleasJ Lab Clin Med 80: 598, 1972. 7. Free, C. H., In Chasin, A. (ed.), Methods in Cyclic Nucleotide Research, Marcel Dekker, New York, 1972, p. 223. 8. Desbuquois, B., and G. D. Aurbach, J Clin Endocrinol Metab 33: 732, 1971. 9. Murphy, B. E. P., J Clin Endocrinol Metab 27: 973, 1967. 10. Furtk J., K. H. Clifton, E. L. Gadsden, and R. F. Buffer, Cancer Res 16: 608, 1956. 11. Milkovic, S., M. M. Garrison and R. W. Bates, Endocrinology 75: 670, 1964. 12. Samuels, H. H., Jr., S. Tsai, and R. Cintron, Science 181: 1253, 1973. 13. Nicoll, C. S., and J. Meites, Endocrinology 72: 544, 1962. 14. Schooley, R. A., S. Friedkin, and E. S. Evans, Endocrinology 79: 1053, 1966. 15. Sayers, G., R. L. Swallow, and N. D. Giordano, Endocrinology 88: 1063, 1971.
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Y
ABSTRACT. The transplantable rat pituitary tumor MtT-F4 failed to grow in rats hypophysectomized at the time of transplantation, but did grow in thyroxine-treated hypophysectomized rats. In the latter rats, the tumor did not stimulate the adrenals to the same extent as in control rats. When the tumor cells were isolated and incubated in vitro, those from hypophysectomized thyroxine-treated rats released much less ACTH into the incubation medium than the tumor cells from control rats. They
also released significantly less growth hormone than tumor cells from intact, intact thyroxine-treated, and thyroidectomized thyroxine-treated rats. Prolactin release by the isolated tumor cells in vitro was the same in all groups studied. The results suggest that the hypophysectomy and thyroxine treatment of the host rat might selectively influence the production of hormones by the MtT-F4 transplantable rat pituitary tumor. (Endocrinology 96: 739, 1975)
'RANSPLANT/ABLE pituitary tumors hormone (5) we determined the production developed by Furth (1) offer a means of these hormones by tumors grown in of studying the secretion and biosynthesis hypophysectomized and thyroxine-treated of pituitary hormones. The secretion of rats in order to compare them with the pituitary hormones by tumor cells differs, endocrine properties of tumors grown in however, from that of normal pituitary intact rats. since the tumor lacks the hypothalamic control that is essential for normal pituitary Materials and Methods function (2). On the other hand, transRats bearing MtT-F4 tumors were obtained planted tumors and their endocrine ac- from Dr. A. E. Bogden, Mason Research Institivities are not entirely free from external tute, Worcester, Massachusetts, who performed influence. For example, estradiol stimu- the first passage from frozen tumors into 100 g lates prolactin production by tumors while female Fischer 344 rats. Before these tumors inhibiting their effect on the enlargement were used for our experiments they were transof adrenals (3). The AtT-20 type of mouse planted again at least 6 more times. For transthe tumor was cut into pieces about pituitary tumor responds to glucocorticoid plantation, 3 1-2 mm across and suspended and washed in treatment by lowering ACTH production (4). medium 199 (Grand Island Biological ComWe have found that the MtT-F4 type of pany). The tumor fragments were then introtransplantable pituitary tumor fails to duced subcutaneously under light ether anesgrow in rats, hypophysectomized at the thesia through a small incision in the lateral time of transplantation, at least during the abdominal region. In experimental groups to be first 30 days after transplantation; tumor compared, all the tumors originated from the growth is restored by injections of transplanted fragments of one tumor. HypoL-thyroxine. As the MtT-F4 tumor is known physectomy (by the transauricular approach), to produce ACTH, prolactin, and growth ovariectomy, thyroidectomy and adrenaly P
Received July 3, 1974. Work supported by grant 4881 of the Medical Research Council of Canada and the National Cancer Institute of Canada.
ectomy were performed under ether anesthesia. Controls were sham-operated. Completeness of hypophysectomy was verified at autopsy. L-thyroxine (Sigma) was injected subcutaneously in a dose of 10 /xg per rat per day. 739
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Endo • 1975 Vol 96 • No 3
LIS, GUERINOT AND CHRETIEN
740
The tumor cells were isolated by collagenase digestion (M. Lis and M. Chretien, manuscript in preparation). Animals were killed by decapitation and tumors were immediately dissected into pieces about 1-2 mm3 across, washed in saline, and incubated in 10 ml of Ringer-Krebs bicarbonate-glucose (1 mg glucose per ml) containing 30 mg of collagenase (Worthington). After 15 min of incubation at 37 C with 5% CO 2 -95% O2 as a gas phase, 10 ml of RingerKrebs bicarbonate-glucose containing 10 mg of bovine serum albumin (Fraction V, Pentex), 40 fxg of DNase (Sigma), and 20 /u-g of RNase (Sigma) was added. After a further 15 minutes' incubation the tissue fragments were washed and the cells mechanically dispersed in ice-cold Ringer-Krebs bicarbonate-glucose. The cells were washed twice with 50 ml of the same buffer and once with incubation medium. Incubation medium contained bovine serum albumin (2 mg per ml) and lima bean trypsin inhibitor (Worthington) (0.2 mg per ml) in a mixture of Ringer-Krebs bicarbonate-glucose and synthetic medium 199 in the proportions 4:1. In all experiments the cells were isolated and subsequently incubated in Teflon as was recommended by Sayers et al. (15) for isolated adrenal cells. The integrity of cell membranes of incubated cells was established by the exclusion of trypan blue dye. The concentration of cells was determined by means of a hemocytometer and by the measurement of DNA using the ethydium bromide fluorescence method (6). ACTH was determined in the incubation medium by means of a bioassay using isolated rat adrenal cells in vitro essentially as described by Free (7). As ACTH
standard we used synthetic 1-24 peptide (Cosyntropin, Organon, lot #26153). Prolactin and growth hormone were determined in the incubation medium by a radioimmunoassay using rat prolactin and rat growth hormone radioimmunoassay kits distributed by NIAMD. The prolactin and growth hormone were assayed essentially as described in the recommendations accompanying both radioimmunoassay kits. As the iodinated antigens NIAMD-rat prolactin-I-1 was used for prolactin radioimmunoassay, and NIAMD-rat GH-I-2 for growth hormone radioimmunoassay. As reference preparations we used NIAMD-rat prolactin-RP-1 and NIAMD-rat GH-RP-1, respectively. In the prolactin assay we used a double antibody method. In the growth hormone assay we precipitated the antigen-antibody complex with polyethylene-glycol (8). Corticosterone in rat plasma was determined by a protein-binding method essentially as described by Murphy (9).
Results
Table 1 shows that hypophysectomy and thyroidectomy completely prevented the growth of transplanted MtT-F4 tumors, ovariectomy has no effect and adrenalectomy stimulated growth. The weight of adrenals was increased in tumor-bearing rats with corresponding higher corticosterone concentrations in plasma. Thymus weight was decreased by tumor in the presence of adrenals. In adrenalectomized animals, however, the tumor seemed to increase thymus weight.
TABLE 1. Observations on rats with transplanted MtT-F4 tumors Weight Tumor (g)
Control 1.66 ±0.30
Hypophysectomized
Thyroidectomized
Ovariectomized
Adrenalectomized
0
0
2.55 ±0.41
9.4* ±3.4
Adrenals (mg)
171.0 ± 17.7
17.4** ±1.6
Thymus (mg)
44.6 ±6.8
200.0** ±18.7
Corticosterone /i.g/100 ml plasma
45.0 ±4.9
4.15** ±1.24
27.5** ±2.8 95.0* ±14.9 14.4** ±4.6
174 ±22
0
23.5 ±4.7
398** ±29
59.7* ±2.6
2.7** ±2.4
The rats were killed 30 days after tumor transplantation. All data are the means ± standard error. There were 12 rats in each group. Significantly different results, as compared to control group, are indicated by *(95%) and **(99%).
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MtT-F4 TUMOR AFTER HYPOPHYSECTOMY
741
TABLE 2. Effect of L-thyroxine injection on rats with transplanted MtT)F4 tumors Weight
Hypophysectomized
Control
Tumor (g)
162 ±21
Thymus (mg)
81.4 ± 14.3
Thyroidectomized + T4
1.20 ±0.37
1.52 ±0.19
0
1.77 ±0.48
Adrenals (mg)
Hypophysectomized + T4
35.5** ±5.4
12.8** ± 1.0 184** ± 16
236** ±20
119 ±22 92.1 ±17.5
Conditions and statistics as in Table 1 (but separate experiment). T4 = L-thyroxine, 10 /xg/day SC for 30 days.
Daily administration of 10 fig L-thyroxine (T4) permitted tumor growth in hypophysectomized animals (Table 2), but the adrenals were not stimulated and the thymus did not regress as in the control tumorbearing rats. In the thyroidectomized T4treated rats, tumors grew well, the adrenals 0.3r
were stimulated, and the thymus regressed. This difference suggests a decreased ACTH production by tumor tissue in the hypophysectomized groups. This was confirmed by incubation of the tumor cells isolated from these rats and measurement of the ACTH released (Fig. 1). We also found (Fig. 2) that isolated tumor cells from thyroxine-treated, hypophysectomized rats released substantially less growth hormone than did corresponding cells from the other three groups, and this accords well with the substantially lower plasma concentration of growth hormone in these animals (Table 3). The amounts of
* - *
TUMOR CELLS M I T - F j i H p i ; T«
O—6 TUMOR CELLS MIT-F4; Ttii; T/| •—•
TUMOR CELLS MIT-F4; T4
O - O TUMOR CELLS MIT-F*
TUMOR CELLS M»T-F 4 :Hp«:T4
2
4
TIME OF INCUBATION (hours)
FIG. 1. ACTH release by isolated tumor cells. The isolated cells from 4 tumors from different rats in each group were incubated in the medium described under Materials and Methods and incubation medium was collected at the intervals indicated. Aliquots of this medium were then incubated with isolated rat adrenal cells, and corticosterone production was related (see ref. 7) to ACTH standards (synthetic 1-24). All values are the means of two measurements. The experiment was repeated with essentially the same results. Hpx; T4) hypophysectomized host rats treated with L-thyroxine.
TIME OF INCUBATION (hours)
FIG. 2. Growth hormone released by isolated tumor cells. In each incubation the isolated cells from 2 tumors were used. Growth hormone was measured in the incubation medium by radioimmunoassay. All measurements are duplicates and the results are the means of 4 different incubations ± standard error. Hpx, hypophysectomy; T4, L-thyroxine treatment; Thx, thyroidectomy.
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Endo • 1975 Vol 96 • No 3
LIS, GUERINOT AND CHRETIEN
742
prolactin released by the tumor cells from
inhibited tumor growth (rat MtT-F4) and the inhibition was prevented by thyroxine. In our experiments there was no palpable MtT-F4 growth for 30 days after transplantation in thyroidectomized or hypophysectomized rats, while all other groups produced tumors weighing more than 1 g. The difference may lie in the fact that we performed the operations and started thyroxine treatment at the time of transplantation. Mizuno et al. (3) started the thyroxine or thiouracil treatment as late as 4-5 weeks after transplantation. An in vitro stimulating action of thyroxine and L-triiodothyronine on glucose utilization by
all rats were the same (Fig. 3) as were the
cultured pituitary tumor (GH, clone) has
TABLE 3. Plasma growth hormone concentrations of tumor-bearing rats Group Tumor Tumor + T4 Tumor + Hpx + T4 Tumor + Thx + T4
GH (ng/ml plasma) 205 ± 255 ± 144 ± 208 ±
15 11 11* 21
(7) (8) (8) (7)
* Significantly different (P < 0.05). Blood samples were collected under light ether narcosis from jugular vein 26 days after tumor transplantation. GH was measured by radioimmunoassay. The plasma concentrations were correlated to lg of tumor weight. Means ± SE (number of observations in parenthesis).
plasma prolactin concentrations (not shown).
recently been reported (12). The thyroid hormones can influence normal pituitaries: for example, thyroxine stimulates prolactin Discussion release by rat anterior pituitaries in vitro (13). Moreover, thyroidectomy caused a Transplantable mammotropic pituitary gradual decrease in the population of tumors were developed in rats by Furth somatotrophic cells (14). The effect reand coworkers (10). The MtT-F4 line of ported in our experiments does not seem to transplantable pituitary tumor was dea simple effect of thyroxine treatment. be veloped in rats by the implantation of The treatment of intact tumor-bearing rats stilbestrol pellets. At first, these tumors grew only in estrogen-treated hosts, but with thyroxine, or the treatment of subpassaging produced an autonomous var- thyroidectomized tumor-bearing rats with iant which grows in intact Fischer rats (10). thyroxine did not cause any detectable The MtT-F4 tumors were reported to changes when compared with controls (Tasecrete ACTH, prolactin, and growth hor- bles 2 and 3, Fig. 2). Thyroxine treatment mone and to increase plasma concentrations of these substances 6000 times, 50 A — A TUMOR CELLS MtT-F« , Hpi, T4 times, and 30 times, respectively (5). AlA — A TUMOR CELLS MIT-F4 ;TI>« j T • — • TUMOR CELLS MlT-F ;T though the tumors grew autonomously, O — O TUMOR CELLS MlT-F several workers noted that changing the hormonal balance of host rats or of the incubation medium could influence the growth and endocrine properties of these tumors. For example, estradiol, cortisol, and thyroxine treatment of rat bearing MtT-F4 tumors raised plasma prolactin concentrations (3). There are, however, some discrepancies between our results and those of others. For example Mizuno et TIME OF INCUBATION (hours) al. (3) reported no influence of thyroxine or thiouracil on MtT-F4 tumor growth, while FIG. 3. Prolactin released by isolated tumor cells. was measured by radioimmunoassay in the Milkovic et al. (11) found that thyroidec- Prolactin same incubation medium samples as were used for tomy or thiouracil treatment significantly Fig. 2. 4
4
4
4
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MtT-F4 TUMOR AFTER HYPOPHYSECTOMY was sufficient to restore tumor growth in hypophysectomized animals, but apparently failed to produce the usual endocrine profile of tumors grown in intact hosts. The nature of the factor(s) required for the maintenance of the usual endocrine pattern of MtT-F4 tumors remains to be elucidated. Acknowledgments The authors thank Mrs. F. Rousseau for her excellent technical assistance. The secretarial help of Mrs. D. Marcil is also appreciated. The authors are indebted to NIAMD for providing the radioimmunoassay kits for rat prolactin and rat growth hormone, to Dr. A. E. Bogden, Mason Research Institute for MtT-F4 tumor, and to Organon Inc. for the gift of Cosyntropin peptide.
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