BrIef CommunIcatIon: In Vitro Stimulation of Human Breast Tissue by Human Prolactin
1,2
W. G. Dilley 3 and S. J. Kister 4,5
Mitotic
SUMMARY-Normal human breast tissue was cultured with defined media plus hormones. The epithelium survived at least 4 days in culture but did not grow in the absence of hormones. Both Insulin and human prolactin stimulated growth, but ovine prolactin did not.-J Natl Cancer Inst 55: 35-36, 1975.
MAMMOGENIC HORMONES in the normal human female have not yet been precisely defined (1). Recent reports that prolactin-releasing drugs derived from Rauwolfia sp. are related to a greatly increased risk of breast cancer (2-4) focused attention on the potential activities of prolactin on human breast tissue. Several recent studies indicated a positive response of human breast tissue to ovine prolactin (oProl) or bovine prolactin in vitro (5-8). In the present investigation, the growth-promoting effects of insulin, oProl, and human prolactin (hProl) on normal human breast tissue in organ culture are described.
Ind••
lA•
f-
1.2
~
1.0
RESULTS
The principal results are presented in text-figure 1. The colchicine and 3H-TDR results were combined: the colchicine and tritium percentages on the basis of 8 and 2 hours of incubation, respectively. There was little mitosis or DNA synthesis in the absence of hormones in vitro; however, the tissue appeared healthy. The addition of I caused an increase of 390% above the NH group. I-oProl produced only a minimal increase above that obtained with I alone. However, I-hProl or I-HPL resulted in a 36% increase above that of the group with only I. The Student's t-test yielded a value less than 0.1 for the comparison of the I group versus the I-oProl group. DISCUSSION
The results indicate a highly significant increase in mitotic activity when I is added to the culture medium.
-
,..
-
.8 .6 .4
MATERIALS AND METHODS
Biopsy specimens of normal human breast tissue were obtained from 30 women suspected of having breast cancer; most had benign breast disease. Each specimen was cut into about fifty explants that were distributed into various hormonal media groups (results of all the groups will be reported in a full-length article). Media consisted of Waymouth's MB 752/1 with no hormones (NH), insulin (I), I plus ovine prolactin (I-oProl), or I plus human prolactin (I-hProl). In some cases, human placental lactogen (HPL) was substituted for h'Prol. All hormones were used at 5p.g/ml. Explants were incubated in Falcon organ culture dishes (Falcon Plastics, Oxnard, Calif.) on Dacron rafts at 37° C for 4 days in an atmosphere of 5% CO2:55% O 2:40% N 2 • Eight hours before termination, 0.1 p.g colchicine/ml or 1 p.Ci tritiated thymidine (3H-TDR) /ml was added to each culture. Mitotic figures or labeled cells were counted in the histologic sections. Identifying labels were always masked to prevent bias. Two thousand cells were counted from each culture.
-
.2
-
--
......, N
% Z
-co
N
-
-" ~
on
~
'0
0
Go
Go
.!.
.!.
~
0
~
~
TEXT-FIGURE I,-Growth effects of hormones on breast tissue in vitro. Mitotic indices are averaged responses (No. of cultures shown in parentheses) to hormones at the end of 4-day cultures. Averages are percent mitotic figures or percent labeled cells corrected to a basis of an 8-hour colchicine or 2-hour 3H-TDR incubation. Bar labeled I-hProl represents explants cultured in either I-hProl or I-HPL.
The further addition of oProl caused only a minimal increase over that obtained with I alone. However, the hProl (or HPL) increased mitotic activity, significant at the 10% level. This may not seem adequate, but, other experiments comparing oProl and hProl combined with other hormones fully corroborate this difference between hProl and oProi. These experiments have been submitted for publication in a full-length article. The question of whether prolactin acts in the absence of I has been examined in rat and mouse mammary-organ cultures, and I has been necessary for the action of other hormones (9, 10) . However, we are currently testing the relationship of prolactin to lover a broad concentration range on human breast tissue. Prolactin has been implicated as one of the principal factors in murine tumors induced by mammary tumor Received' February 4, 1975; accepted March 21, 1975. Supported by Public Health Service grant CAI3696 from the National Cancer Institute. 3 Department of Anatomy, College of Physicians and Surgeons, Columbia University, New York, N.Y. 10032. 4 Department of Surgery, College of Physicians and Surgeons, Columbia University. 5 We thank Dr, Henry Friesen, Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada; Eli Lilly and Co., Indianapolis, Ind.: and the National Institutes of Health Hormone Distribution Program, Bethesda, Md. for providing the human prolactin, bovine insulin, and human placental lactogen and ovine prolactin, respectively. 1
2
JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 55, NO. I, JULY 1975
35
36
DILLEY AND KISTER
virus (11) and also in neoplasms induced in rats by chemical carcinogens (12). The recent reports of reserpine-related risk for human breast cancer (2-4) suggest a prominent function of prolactin in human carcinogeneSIS.
Most previous reports of in vitro effect of prolactin on normal or malignant human breast tissue have specified either oProl or bovine prolactin. The lack of activity of nonsimian growth hormones in vivo in humans is well known (13). Bovine prolactin and oProl preparations were claimed to be lactogenic in women (14). However, these preparations were crude and probably contained other factors that influence lactation. In any case, the early reports have not been substantiated with purified preparations of nonsimian prolactin. The present study suggests that maximal effects on normal human breast tissue can be obtained with human hormones; further studies to specify the carcinogenic function of prolactin in vitro on human tissue should utilize human prolactin. The importance of this specificity is suggested by recent results in which the differences between oProl and hProl are greatly magnified when they act on human breast cancer (Dilley WG, Kister 8J: In preparation) . REFERENCES (1) LLOYD CW: The Ovaries. In Textbook of Endocrinology, 4th ed (Williams RR, ed.). Philadelphia, WB Saunders Co., 1968, p 459-536 (2) BOSTON COLLABORATIVE DRUG SURVEILLANCE PROGRAM: Reserpine and breast cancer. Lancet 2:669-671, 1974
(3) ARMSTRONG B, STEVENS N, DOLL R: Retrospective study of the association between use of Rauwolfia derivatives and breast cancer in English women. Lancet 2:672-{j75, 1974 (4) HEINONEN OP, SHAPIRO S, TUOMINEN L. et al: Reserpine use in relation to breast cancer. Lancet 2:675-677, 1974 (5) CERIANI RL, CONTESSO GP: Hormone requirements for growth and differentiation of the human mammary gland in organ culture. Cancer Res 32:2190-2196, 1972 (6) SALIH H, BRANDER W, FLOR H: Prolactin dependence in human breast cancers. Lancet 2:1103-1105, 1972 (7) FLAXMAN BA, LASFARGUES EY: Hormone-independent DNA synthesis by epithelial cells of adult human mammary gland in organ culture. Proc Soc Exp Biol Med 143:371374, 1973 (8) LAGIOS MD: Hormonally enhanced proliferation of human breast cancer in organ culture. An in vitro system for assessment of specific hormonal response. Oncology 29:2233, 1974 (9) ICHINOSE RR, NANDI S: Influence of hormones on lobuloalveolar differentiation of mouse mammary glands in vitro. J Endocrinol 35:331-340, 1966 (10) DILLEY \VG: Hormone-dependent differentiation of rat mammary glands in vitro. M.A. Thesis, University of California, Berkeley, 1965 (11) BERN HA, NANDI S: Recent studies of the hormonal influence in mouse mammary tumorigenesis. Prog Exp Tumor Res 2:90-144, 1961 (12) MEITES J: Relation of prolactin and estrogen to mammary tumorigenesis in the rat. J Natl Cancer Inst 48:1217-1224, 1972 (lJ) KNOBEL E, GREEP RO: The physiology of growth hormone with particular reference to its action in the Rhesus monkey and the "species specificity" problem. Recent Prog Horm Res 15:1-53, 1959 (14) KENNY M, KLING EJ: Effect of prolactin on lactation in nursing women. Lancet 2:828-830,1939
1,2
W. G. Dilley 3 and S. J. Kister 4,5
Mitotic
SUMMARY-Normal human breast tissue was cultured with defined media plus hormones. The epithelium survived at least 4 days in culture but did not grow in the absence of hormones. Both Insulin and human prolactin stimulated growth, but ovine prolactin did not.-J Natl Cancer Inst 55: 35-36, 1975.
MAMMOGENIC HORMONES in the normal human female have not yet been precisely defined (1). Recent reports that prolactin-releasing drugs derived from Rauwolfia sp. are related to a greatly increased risk of breast cancer (2-4) focused attention on the potential activities of prolactin on human breast tissue. Several recent studies indicated a positive response of human breast tissue to ovine prolactin (oProl) or bovine prolactin in vitro (5-8). In the present investigation, the growth-promoting effects of insulin, oProl, and human prolactin (hProl) on normal human breast tissue in organ culture are described.
Ind••
lA•
f-
1.2
~
1.0
RESULTS
The principal results are presented in text-figure 1. The colchicine and 3H-TDR results were combined: the colchicine and tritium percentages on the basis of 8 and 2 hours of incubation, respectively. There was little mitosis or DNA synthesis in the absence of hormones in vitro; however, the tissue appeared healthy. The addition of I caused an increase of 390% above the NH group. I-oProl produced only a minimal increase above that obtained with I alone. However, I-hProl or I-HPL resulted in a 36% increase above that of the group with only I. The Student's t-test yielded a value less than 0.1 for the comparison of the I group versus the I-oProl group. DISCUSSION
The results indicate a highly significant increase in mitotic activity when I is added to the culture medium.
-
,..
-
.8 .6 .4
MATERIALS AND METHODS
Biopsy specimens of normal human breast tissue were obtained from 30 women suspected of having breast cancer; most had benign breast disease. Each specimen was cut into about fifty explants that were distributed into various hormonal media groups (results of all the groups will be reported in a full-length article). Media consisted of Waymouth's MB 752/1 with no hormones (NH), insulin (I), I plus ovine prolactin (I-oProl), or I plus human prolactin (I-hProl). In some cases, human placental lactogen (HPL) was substituted for h'Prol. All hormones were used at 5p.g/ml. Explants were incubated in Falcon organ culture dishes (Falcon Plastics, Oxnard, Calif.) on Dacron rafts at 37° C for 4 days in an atmosphere of 5% CO2:55% O 2:40% N 2 • Eight hours before termination, 0.1 p.g colchicine/ml or 1 p.Ci tritiated thymidine (3H-TDR) /ml was added to each culture. Mitotic figures or labeled cells were counted in the histologic sections. Identifying labels were always masked to prevent bias. Two thousand cells were counted from each culture.
-
.2
-
--
......, N
% Z
-co
N
-
-" ~
on
~
'0
0
Go
Go
.!.
.!.
~
0
~
~
TEXT-FIGURE I,-Growth effects of hormones on breast tissue in vitro. Mitotic indices are averaged responses (No. of cultures shown in parentheses) to hormones at the end of 4-day cultures. Averages are percent mitotic figures or percent labeled cells corrected to a basis of an 8-hour colchicine or 2-hour 3H-TDR incubation. Bar labeled I-hProl represents explants cultured in either I-hProl or I-HPL.
The further addition of oProl caused only a minimal increase over that obtained with I alone. However, the hProl (or HPL) increased mitotic activity, significant at the 10% level. This may not seem adequate, but, other experiments comparing oProl and hProl combined with other hormones fully corroborate this difference between hProl and oProi. These experiments have been submitted for publication in a full-length article. The question of whether prolactin acts in the absence of I has been examined in rat and mouse mammary-organ cultures, and I has been necessary for the action of other hormones (9, 10) . However, we are currently testing the relationship of prolactin to lover a broad concentration range on human breast tissue. Prolactin has been implicated as one of the principal factors in murine tumors induced by mammary tumor Received' February 4, 1975; accepted March 21, 1975. Supported by Public Health Service grant CAI3696 from the National Cancer Institute. 3 Department of Anatomy, College of Physicians and Surgeons, Columbia University, New York, N.Y. 10032. 4 Department of Surgery, College of Physicians and Surgeons, Columbia University. 5 We thank Dr, Henry Friesen, Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada; Eli Lilly and Co., Indianapolis, Ind.: and the National Institutes of Health Hormone Distribution Program, Bethesda, Md. for providing the human prolactin, bovine insulin, and human placental lactogen and ovine prolactin, respectively. 1
2
JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 55, NO. I, JULY 1975
35
36
DILLEY AND KISTER
virus (11) and also in neoplasms induced in rats by chemical carcinogens (12). The recent reports of reserpine-related risk for human breast cancer (2-4) suggest a prominent function of prolactin in human carcinogeneSIS.
Most previous reports of in vitro effect of prolactin on normal or malignant human breast tissue have specified either oProl or bovine prolactin. The lack of activity of nonsimian growth hormones in vivo in humans is well known (13). Bovine prolactin and oProl preparations were claimed to be lactogenic in women (14). However, these preparations were crude and probably contained other factors that influence lactation. In any case, the early reports have not been substantiated with purified preparations of nonsimian prolactin. The present study suggests that maximal effects on normal human breast tissue can be obtained with human hormones; further studies to specify the carcinogenic function of prolactin in vitro on human tissue should utilize human prolactin. The importance of this specificity is suggested by recent results in which the differences between oProl and hProl are greatly magnified when they act on human breast cancer (Dilley WG, Kister 8J: In preparation) . REFERENCES (1) LLOYD CW: The Ovaries. In Textbook of Endocrinology, 4th ed (Williams RR, ed.). Philadelphia, WB Saunders Co., 1968, p 459-536 (2) BOSTON COLLABORATIVE DRUG SURVEILLANCE PROGRAM: Reserpine and breast cancer. Lancet 2:669-671, 1974
(3) ARMSTRONG B, STEVENS N, DOLL R: Retrospective study of the association between use of Rauwolfia derivatives and breast cancer in English women. Lancet 2:672-{j75, 1974 (4) HEINONEN OP, SHAPIRO S, TUOMINEN L. et al: Reserpine use in relation to breast cancer. Lancet 2:675-677, 1974 (5) CERIANI RL, CONTESSO GP: Hormone requirements for growth and differentiation of the human mammary gland in organ culture. Cancer Res 32:2190-2196, 1972 (6) SALIH H, BRANDER W, FLOR H: Prolactin dependence in human breast cancers. Lancet 2:1103-1105, 1972 (7) FLAXMAN BA, LASFARGUES EY: Hormone-independent DNA synthesis by epithelial cells of adult human mammary gland in organ culture. Proc Soc Exp Biol Med 143:371374, 1973 (8) LAGIOS MD: Hormonally enhanced proliferation of human breast cancer in organ culture. An in vitro system for assessment of specific hormonal response. Oncology 29:2233, 1974 (9) ICHINOSE RR, NANDI S: Influence of hormones on lobuloalveolar differentiation of mouse mammary glands in vitro. J Endocrinol 35:331-340, 1966 (10) DILLEY \VG: Hormone-dependent differentiation of rat mammary glands in vitro. M.A. Thesis, University of California, Berkeley, 1965 (11) BERN HA, NANDI S: Recent studies of the hormonal influence in mouse mammary tumorigenesis. Prog Exp Tumor Res 2:90-144, 1961 (12) MEITES J: Relation of prolactin and estrogen to mammary tumorigenesis in the rat. J Natl Cancer Inst 48:1217-1224, 1972 (lJ) KNOBEL E, GREEP RO: The physiology of growth hormone with particular reference to its action in the Rhesus monkey and the "species specificity" problem. Recent Prog Horm Res 15:1-53, 1959 (14) KENNY M, KLING EJ: Effect of prolactin on lactation in nursing women. Lancet 2:828-830,1939
