55 (1990) 31-37 Elsevier Scientific Publishers Ireland Ltd.
31
CancerLetters,
Neoplastic transformation and DNA damage of mouse mammary epithelial cells by IV-methyl-N’-nitrosourea in organ culture C.R. Delp, J.S. Tumor
Biology
Treves and M.R. Banerjee
Laboratory,
of Biological
School
Sciences,
University
of Nebraska-Lincoln,
Lincoln,
NE 68588-0342
(U.S.A.)
(Received 16 June 1989) (Revision received 10 September (Accepted 12 September 1990)
1990)
Summary
histopathology. In addition, the epithelial cells in organ
in vitro system was used to the effect of a direct-acting carcinogen
MNU
An appropriate study
on the transformation cells
mary
of mammary
epithelial
transform Mouse
and the cells were
parenchyma-free
inguinal
mammary
syngeneic
female
host
tumors
virgin
were
observed
in
of MNU To assay of MNU
alveolar
nodules
Palpable glands
of
(HANS).
Histo-
pathologic examination of the tissues showed that the tumors were mammary adenocarcinoma. All tumors and hyperplasias were secondarily
transplanted
resulting
in tumors
into syngeneic
and hyperplasias
on day 4 and day 5
are susceptible to the carcinogenic action of the direct-acting carcinogen MNU and that the whole mammary gland culture system offers
appropriate in t&o model for studying mechanism of carcinogenesis induced MNU.
23% of the mice after 3-4 months and an additional 31% showed serially transplantable hyperplastic
DNA were
the by
fat pad of
mice.
injected
using the new nick
culture
an
were disinto the
injected
of
of the mammoperiod. These results demonstrate that the mouse mammary epithelial cells
the mammary cells in mammary glands were
treated with single or multiple doses during various periods of the culture. for neoplastic transformation potential on mammary cells, mammary glands sociated
treated
genie
the ability of N-methyl-N’-nitrosou-
rea (MNU) to organ culture.
damage
translation assay. The most extensiue damage occurred when the glands
in the organ culture of the whole mamgland in vitro. Studies were done to
determine
was also measurable
DNA
culture caused by
animals, of similar
Correspondence to: M.R. Banerjee, Tumor Biology Laboratory, 224 Lyman Hall, University of Nebraska-Lincoln, School of Biological Sciences, Lincoln, NE 68588-0342, U.S.A.
0304-3835/90/$03.50 0 1990 Elsevier Scientific Publishers Published and Printed in Ireland
Keywords: direct-acting carcinogen; Nmethyl-N’-nitrosourea; neoplastic transformation; mouse mammary epithelial cells; organ culture in vitro Introduction Direct-acting (activation-independent) carcinogens possess the property of being able to react with nucleophilic target molecules without being altered by the cell’s metabolic machinery. When DNA is the target molecule, DNA damage and mutagenesis can be the
Ireland I_td
32
result (Gullino, 1975) [S] . N-Nitrosamide compounds, such as MNU, are uniformly direct-acting carcinogens which do not require 1978) [4]. enzymatic activation (Grover, These compounds react readily with the cellular macromolecules such as DNA and protein, as well as sulfhydryl compounds at pH of 6.2-7.0 (Becker, 1982) [2]. Previously, the study of the mechanism of mammary cell transformation by MNU has centered around in vivo models. In an in vivo model, Sukumar et al. (1983) [13] have shown that a point mutation in H-t-as-1 oncogene caused by MNU induces tumors in rat mammary glands. Recently, Miyamoto et al. (1988) [9] have shown that MNU also can induce a high frequency transformation of mouse mammary epithelial cells in serum-free culture in vitro. Although these studies have yielded valuable information as to the mode of action of the direct-acting carcinogens, the specific factors affecting the oncogenic process in the whole mammary gland in vitro remains unknown. Therefore, it is desirable to have an in vitro system which closely mimics the molecular and biochemical events of the mammary gland in vivo. Previously, Iyer and Banerjee (1981) [6] have used the whole mammary gland culture model to demonstrate transformation potential of metabolically activated procarcinogens. Using this in vitro model system, it was observed that DMBA induces nodule-like alveolar lesions (NLAL) in the mammary gland in vitro (Lin et al., 1983) [7]. Although the potential of MNU to interact with the DNA of mammary fat pads has been demonstrated in previous studies, the ability of the carcinogen to cause DNA damage (i.e., scission) has only been postulated (Grover, 1978) [4]. The mouse mammary whole organ culture model offers an appropriate model system for these studies. Thus, the present study was undertaken to determine the ability of MNU to enter the mammary fat pad, cause DNA damage in the isolated whole mammary gland and induce neoplastic transformation of the epithelial cells in a serum-free medium, in vitro.
Materials and Methods Animals
All mice used were of the BALB/c strain obtained from the National Institutes of Health (NIH) approved vendors. The BALB/c strain of mice does not express the endogenous murine mammary tumor virus and has a low occurrence of spontaneous mammary tumors (DeOme et al. 1980) [3]. Animal care was in accordance with standards set by the NIH. Organ culture
Mammary glands were cultured as previously described by Banerjee et al. (1983) [ 11. Briefly, 3-4-week-old BALB/c virgin female mice were primed by daily S.C. injections of progesterone (1 .O mg) and 17fi-estradiol (1.0 pg) from Sigma Chemicals for 9 days prior to culture. On the 10th day the mice were euthanized by cervical dislocation and the whole second thoracic mammary gland, including the primary duct and nipple, were aseptically excised on a Dacron raft. The mammary gland resting on the raft was transferred into a 60 x 15 mm plastic culture dish containing 4 ml of Waymouth’s MB752/2 culmedium (Grand Island Biological ture Company) supplemented with the following steroid and peptide hormones obtained from Sigma, St. Louis, MO; insulin, prolactin and hydrocortisone (5 pg/ml) and aldosterone (1 pg/ml). Penicillin was added at a concentration of 3.5 pg/ml. This medium is hereafter referred to as the mammogenic medium. The glands (4 glands to a culture dish) were placed in a 37OC humidified chamber with an atmosphere of 95% oxygen and 5% carbon dioxide. The media was changed every other day. Approximately 5% of the glands from each culture were stained and microscopically examined for lobulo-aveolar development. The staining procedure was as follows: glands were fixed for 1 h in modified Carnoy’s solution (ethanol and glacial acetic acid 3:l v/v), washed for 15 min in 70% ethanol, rinsed in distilled water, stained for 12 h in alum car-
33
mine, dehydrated in graded concentrations of alcohol and defatted in toluene for microscopic examination. Carcinogen treatment MNU was dissolved in dimethylsulfoxide (DMSO) at a concentration of 33 pg/pl and added to the cultures within 3 min of preparation. In the transplantation assay, the glands were treated twice with a MNU dose of 125 pg/ml medium. Both MNU treatments were each 45 min in duration and occurred once on day 3 and once on day 7 of the culture period. After the 7th day in culture, the glands were retained in the mammogenic media for 6 more days to ensure recovery from the carcinogen treatment. For the nick translation assay, separate cultures received a l-h MNU treatment of 250 pg/ml medium on different days of the mammogenic culture period. Tissue dissociation The tissue dissociation procedure was modified from that described by Richard et al. (1983) [12]. Briefly, MNU treated and DMSO treated (control) mammary glands were removed from the Dacron rafts using sterile forceps and blotted dry on a sterile Whatman qualitative filter. Thirty glands from each experimental group were pooled, placed in a sterile petri dish and weighed. The tissue was minced finely with crossed scalpels until the pieces were less than 1 mm3. The minced tissue was transferred into a trypsinization flask containing a sterile solution of collagenase in the ratio of 1 g tissue : 6 mg collagenase type III (Sigma) : 10 ml medium 199 (Grand Island Biological Company). The flask was shaken at 70 rev./min for 3 h at 37OC. The cell suspension was transferred to sterile centrifuge tubes and centrifuged at 180 X g for 10 min. The supernatant was discarded and the cell pelIet was resuspended and transferred to another flask containing 0.04% DNase and pronase in the ratio of 1 g original tissue : 1 mg pronase : 10 ml medium 199. The flask was shaken at 70 rev./min for 10 min at 37OC. The cell sus-
pension was poured through a sterile gauze filter to remove large cell clumps and fatty tissue. The cells were pelleted at 180 x g for 10 min and resuspended in 1 ml of medium 199. The cells were counted on a hemocytometer and the viability was determined using the Trypan blue exclusion test. Erythrocytes and white blood cells were not counted. The suspensions were discarded, if the cells in clumps could not be counted, or if the viability was less than 80%. Transplantation Three-week-old BALB/c virgin female mice were used as the recipients for the carcinogentreated cells. The host animals were anesthetized with nembutal solution (0.06 mg/g body wt.). Mice under 10 g were not used. The inguinal mammary glands were exposed by a midventral incision. Rudiments of the inguinal mammary gland including host epithelial tissue were removed by cauterization. Using a Hamilton No. 710SN syringe fitted with a 0.5-inch 30 gauge needle, 0.1 ml of the cell suspension (1 x lo5 cells) was slowly injected into the center of the remaining fat pad. In the left inguinal fat pad the MNU treated cells were injected and in the right inguinal fat pad the DMSO treated (control) cells were injected. The animals were then sutured and maintained in separate cages as virgins. The animals were palpated every week for the presence of tumors, and after 4 months all animals not bearing tumors were reopened and examined for the presence of mammary hyperplasia. Tissue thought to be hyperplastic was transplanted into syngeneic host animals to test its tumorigenic potential. This procedure involved placing a small piece (1 mm3) of the abnormal tissue into the cleared inguinal fat pad of a 3-week-old BALB/c virgin female mouse. In addition, small pieces of both hyperplastic and tumorous tissue were fixed in acetic alcohol, dehydrated in graded concentrations of alcohol, embedded in paraffin, sectioned at 5 PM and stained in hematoxylin and eosin for histological examination. After 10 weeks, all
34
mice secondarily transplanted with tissue originally thought to be hyperplastic or tumorous were reopened and examined for mammary outgrowths having the same morphology as the’primary transplanted tissue. Also, small pieces (1 mm3) of the tumorigenie tissue were subcutaneously transplanted onto the dorsal surface of syngeneic host BALB/c virgin female mice. Mice developing subcutaneous tumors were reopened and a small piece of the tumor was taken for histological examination. Nick translation The nick translation assay was based on the procedure developed by Nose and Okamato (1983) [ 111. Mammary cells previously isolated via the tissue dissociation procedure were resuspended in 5 ml of a solution containing 0.25 M sucrose, 0.1 M Tris-HCI (pH 7.4), 10 mM MgCI, and 0.5 mM dithiothreitol. LysoIecithin (100 pg/ml) was added just before use. The cells were kept on ice for 5 min then centrifuged at 180 x g for 10 min. The cell pellet was resuspended in the nick translation assay mixture containing 50 mM Tris-HCI (pH 7.4), 5 mM MgCI,, 10 mM 2-mercaptoethanol, 50 pg/ml bovine serum albumen, 0.05 mM of each dATP, dGTP and dTTP and 5 @/ml [3H]dCTP. E. coli DNA-polymerase I (50 units/ml) were then added and the reaction was allowed to proceed at room temperature for 30 min. After the reaction period, the entire mixture was applied to Whatman-3MM filter disks pre-wetted with 2% pyrophosphate on a Millipore suction filter. The filter disks were extensively washed with 5% trichloroacetic acid containing 1% pyrophosphate and
Table 1.
Neoplastic transformation
of mammary
rinsed with 100% ethanol. After drying, the acid insoluble radioactivity was measured by scintillation spectrometry on a Beckman 1201 liquid scintillation counter. The nick translation assays were conducted immediately after the 1 h carcinogen treatment. Results and Discussion To establish the neoplastic transformation potential of the direct-acting carcinogen on mammary epithelial cells in organ culture of the whole mammary gland in vitro, a transplantation assay was performed. The ability of MNU to induce neoplasm in the host mouse mammary gland was examined by injecting the cells from dissociated MNU treated glands vitro, into virgin female BALB/c host mice mammary fat pads, cleared of host epithelial tissue to prevent competition. The first set of transplantation experiments using cultured mammary glands treated with a single dose of MNU (250 pg/mI) failed to induce tumors or HANS in the glands of syngeneic host animals (Table I). Miyamoto et al. (1988) [9] had reported mammary cell transformation in a cell culture model using two treatments of MNU, so the tumorigenic potential of gland treated twice with MNU in culture was tested. Thirteen animals were used in the transplantation assay to test mammary glands treated twice in culture with MNU (125 pg/ml), once on day 3 and day 7. This carcinogen treatment gave rise to both tumors and HANS. Twentythree percent (3 mice) of the transplanted host mice developed palpable tumors within 3-4 months. An additional 31% (4 mice) of the transplanted host mice showed abnormal
epithelial cells in organ culture.
Experiment
Treatment
No. of fat pads
5%HANS
46 Tumors
I
1 MNU treatment 1 DMSO treatment 2 MNU treatments 2 DMSO treatments
42 42 13 13
0 0 31 0
0 0 23 0
I II II
All animals were successfully transplanted.
Fat pads not containing
HANS or tumors were normal in development.
35
hyperplastic growth. Pieces of all tumors and HANS were taken for histological examination and secondary transplantation. All hyperplasias were alveolar in origin with no ductal hyperplasias observed (Fig. 1). Tumor sections were determined to be adenocarcinoma of mammary epithelial cell origin. Figure 2 shows the invasive cellular extensions of the carcinoma. The secondary transplants all develabnormal oped outgrowths having a morphology and histology similar to their primary outgrowths within 10 weeks after transplantation. None of the parenchyma-free mammary fat pads of the host mice transplanted with cells from the dissociated DMSO treated (control) glands developed abnormal tissue. The final test for true adenocarcinoma is growth of the tumor at an ectopic site. Small
Fig. 2. Histological section gland showing the invasiveness induced try MNU.
Fig. 1. mammary lar origin.
Histological section of MNU treated mouse gland showing that the hyperplasia is of alveo-
of a mouse mammary of the adenocarcinoma
pieces of the tumorous tissue were S.C. transplanted onto the backs of 12 host BALB/c mice. Sixty-seven percent (8 mice) of the S.C. transplanted mice developed adenocarcinomas. Mouse mammary carcinogenesis in vivo is associated with the appearance of the HANS, followed by mammary tumors which appear later in the hyperplasia. Studies have shown that HAN tissue is serially transplantable into the parenchyma-free mammary fat pad of syngeneic mice. Prior to the tissue dissociation, MNU treated glands from each day of the 7 days mammogenie period, in culture, were placed separately in regression media (Waymouth’s MB752/2 culture medium supplemented with only insulin and penicillin) for 15 days and stained as previously described. Stained glands were assayed microscopically for the presence
36
of NLAL representing foci of cells which have escaped from normal hormonal control and remained unregressed indicating damage caused by the carcinogenic treatment (Fig. 3). Studies in our laboratory have demonstrated that the chemical carcinogen 7,12dimethylbenz[a]anthracene (DMBA) can induce neoplastic transformation of the mammary cells in a serum-free hormonally defined culture medium (Iyer and Banerjee, 1981) [6]. Additional studies have shown that the neoplastic transformation of the mammary gland in vitro is associated with the appearance of NLAL which, like HAN, are serially transplantable into the parenchyma free mammary fat pad of syngeneic female mice. Since the appearance of HAN and NLAL precedes the tumor formation, HAN and NLAL are designated as precancerous and HAN and NLAL
are related to neoplastic transformations. In this respect the neoplastic transformations induced by MNU and by DMBA appear to be rather similar and the studies described in this paper clearly indicate that the direct acting carcinogen MNU can induce neoplastic transformation of the mammary cells in organ culture. At present there is no report of a DNA damage study showing that the neoplastic transformation of the mammary cells induced by MNU can be caused by DNA damage in the mammary cells in vitro. Although an earlier study has reported that the direct acting carcinogen MNU can induce a high frequency of sister chromatid exchange (SCE). But the high frequency of SCE induced by MNU represents chromosomal rearrangements rather than DNA damage. This deficiency provided the rationale for including the quantitative measurement of DNA damage using the nick-translation assay. DNA damage was observed during all 6 days of the mammogenic period, with the greatest damage occurring between day 4 and day 5 of culture (Table II). Explants from control glands treated with DMSO consistently
Table II. MN&induced DNA damage as measured by nick-translation assay in mouse mammary epithelial cells during different stages of mammary development. Day treated
Treatment
Incorporation cpm/105 cells*
1 2
MNU DMSO MNU DMSO MNU DMSO MNU DMSO MNU DMSO
3840 610 6900 425 7204 349 7555 699 6035 561
3 3 4 4
Fig. 3. Wholemount of a stained mouse mammary gland treated with MNU showing the presence of nodulelike alveolar lesions (NLAL) Arrow indicates NLAL.
5 5 6 6
‘Each value is the mean of 3 separate different batches of glands.
f f & f f + * + f f
32 29 25 56 55 60 45 55 87 45
experiments
with
37
showed a basal level of DNA damage, as evidenced by a lower incorporation of [3H]deoxycytidylate. This low level of radioactivity indicates a basal level of DNA damage occurring within the cell. In cells treated with the carcinogen, the level of incorporation was at least lo-fold greater than in the control cells treated with DMSO. Consideration of the findings discussed in the preceding paragraphs reiterate the concept that components of the intact cells interact with the direct-acting carcinogens thus inducing the neoplastic transformation of the mammary epithelial cells. This observation is consistent with the possibility that MNU can functionally activate the expression of the endogenous oncocells. Recently, genes in the mammary Miyamoto et al. (1990) [lo] have indicated that mouse mammary epithelial cells transformed in primary culture with MNU have a specific mutation in the c-Ki-ras proto-oncogene and that this mutation is a preneoplastic event in mouse mammary carcinogenesis induced by MNU.
References 1
2 3
4 5
6
7
8
9
Acknowledgements We thank Dr. Sanjoy K. Das and Dr. Abhik M. Bandyopadhyay for their comments and discussion during preparation of the manuscript, Michelle Mathiesen for her technical assistance, Robert Schmaltz for editing assistance, Rodney S. Markin, M.D., Ph.D., Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, for identification of the pathological type of the tumors and Donna M. McCarthy for her secretarial assistance. This supported by Grant investigation was CA25304 awarded by the National Cancer Institutes. USPHS.
10
11
12
13
Banerjee, M.R. (1983) In: In Vitro Model for Cancer Research. Editors: M. Weber and L. Sekely. CRC Press, Boca Raton. Becker, F.F. ed. (1982) Cancer 1, a comprehensive treatise. 2nd edn., p. 263. Plenum Press, New York. DeOme, K.B., Osborne, R.C. and Miyamoto, M.J. (1980) In: Cell Biology of Breast Cancer, p. 79. Editors: C.C. McGrath, M. Brennan and M. Rich. Academic Press, New York. Grover, P.L. (1978) Chemical carcinogens and DNA. CRC Press, Boca Raton. Gullino, P. (1975) N-nitrosomethylurea as mammary gland carcinogen in rats. J. Natl. Cancer Inst., 54, 401411. lyer, A. and Banerjee, M. (1981) Sequential expression of preneoplastic and neoplastic characteristics of mouse mammary epithelial cells transformed in organ culture. J. Natl. Cancer Inst., 66,893-899. Lin, F.K., Banerjee, M.R. and Crump, R.L. (1976) Cell cycle-related hormone-carcinogen interaction during chemical carcinogen induction of NLAL in organ culture. Cancer Res., 36, 1607. Manoharan, K. and Banerjee, M.R. (1985) Measurements of chemical carcinogen-induced sister-chromatid exchanges in a whole organ in vitro. Mutat. Res., 147, 165- 169. Miyamoto, S., Guzman, R., Osborne, R. and Nandi, S. (1988) Neoplastic transformation of mouse mammary epithelial cells by in vitro exposure to N-methyl-N-nitrosourea. Proc. Natl. Acad. Sci. USA, 85,477-481. Miyamoto, S., Sukumar, S., Guzman, R.C., Osborn, R.C. and Nandi, S. (1990), Transforming c-Ki-ras mutation is a preneoplasttc event in mouse mammary carcinogenesis induced in vitro by N-methyl-N-nitrosourea. Mol. Cell. Biol., 10, 1593-1599. Nose, K. and Okamato, H. (1983) Detection of carcinogen-induced DNA damage by nick translation in permeable cells. Biochem. Biophys. Res. Commun., 111,383. Richard, J.D., Pasco, R., Guzman, R. and Nandi, S. (1983) Comparison for the growth of normal and neoplastic mouse mammary cells on plastic and collagen gels. Exp. Cell Res., 146, l-14. Sukumar, S., Notario, V., Martin-Zanca, D. and Barbacid, M. (1983) Induction of mammary carcinomas in rats by MNU involves malignant activation of H-ras-1 locus by single point mutations. Nature, 306,658-661.
31
CancerLetters,
Neoplastic transformation and DNA damage of mouse mammary epithelial cells by IV-methyl-N’-nitrosourea in organ culture C.R. Delp, J.S. Tumor
Biology
Treves and M.R. Banerjee
Laboratory,
of Biological
School
Sciences,
University
of Nebraska-Lincoln,
Lincoln,
NE 68588-0342
(U.S.A.)
(Received 16 June 1989) (Revision received 10 September (Accepted 12 September 1990)
1990)
Summary
histopathology. In addition, the epithelial cells in organ
in vitro system was used to the effect of a direct-acting carcinogen
MNU
An appropriate study
on the transformation cells
mary
of mammary
epithelial
transform Mouse
and the cells were
parenchyma-free
inguinal
mammary
syngeneic
female
host
tumors
virgin
were
observed
in
of MNU To assay of MNU
alveolar
nodules
Palpable glands
of
(HANS).
Histo-
pathologic examination of the tissues showed that the tumors were mammary adenocarcinoma. All tumors and hyperplasias were secondarily
transplanted
resulting
in tumors
into syngeneic
and hyperplasias
on day 4 and day 5
are susceptible to the carcinogenic action of the direct-acting carcinogen MNU and that the whole mammary gland culture system offers
appropriate in t&o model for studying mechanism of carcinogenesis induced MNU.
23% of the mice after 3-4 months and an additional 31% showed serially transplantable hyperplastic
DNA were
the by
fat pad of
mice.
injected
using the new nick
culture
an
were disinto the
injected
of
of the mammoperiod. These results demonstrate that the mouse mammary epithelial cells
the mammary cells in mammary glands were
treated with single or multiple doses during various periods of the culture. for neoplastic transformation potential on mammary cells, mammary glands sociated
treated
genie
the ability of N-methyl-N’-nitrosou-
rea (MNU) to organ culture.
damage
translation assay. The most extensiue damage occurred when the glands
in the organ culture of the whole mamgland in vitro. Studies were done to
determine
was also measurable
DNA
culture caused by
animals, of similar
Correspondence to: M.R. Banerjee, Tumor Biology Laboratory, 224 Lyman Hall, University of Nebraska-Lincoln, School of Biological Sciences, Lincoln, NE 68588-0342, U.S.A.
0304-3835/90/$03.50 0 1990 Elsevier Scientific Publishers Published and Printed in Ireland
Keywords: direct-acting carcinogen; Nmethyl-N’-nitrosourea; neoplastic transformation; mouse mammary epithelial cells; organ culture in vitro Introduction Direct-acting (activation-independent) carcinogens possess the property of being able to react with nucleophilic target molecules without being altered by the cell’s metabolic machinery. When DNA is the target molecule, DNA damage and mutagenesis can be the
Ireland I_td
32
result (Gullino, 1975) [S] . N-Nitrosamide compounds, such as MNU, are uniformly direct-acting carcinogens which do not require 1978) [4]. enzymatic activation (Grover, These compounds react readily with the cellular macromolecules such as DNA and protein, as well as sulfhydryl compounds at pH of 6.2-7.0 (Becker, 1982) [2]. Previously, the study of the mechanism of mammary cell transformation by MNU has centered around in vivo models. In an in vivo model, Sukumar et al. (1983) [13] have shown that a point mutation in H-t-as-1 oncogene caused by MNU induces tumors in rat mammary glands. Recently, Miyamoto et al. (1988) [9] have shown that MNU also can induce a high frequency transformation of mouse mammary epithelial cells in serum-free culture in vitro. Although these studies have yielded valuable information as to the mode of action of the direct-acting carcinogens, the specific factors affecting the oncogenic process in the whole mammary gland in vitro remains unknown. Therefore, it is desirable to have an in vitro system which closely mimics the molecular and biochemical events of the mammary gland in vivo. Previously, Iyer and Banerjee (1981) [6] have used the whole mammary gland culture model to demonstrate transformation potential of metabolically activated procarcinogens. Using this in vitro model system, it was observed that DMBA induces nodule-like alveolar lesions (NLAL) in the mammary gland in vitro (Lin et al., 1983) [7]. Although the potential of MNU to interact with the DNA of mammary fat pads has been demonstrated in previous studies, the ability of the carcinogen to cause DNA damage (i.e., scission) has only been postulated (Grover, 1978) [4]. The mouse mammary whole organ culture model offers an appropriate model system for these studies. Thus, the present study was undertaken to determine the ability of MNU to enter the mammary fat pad, cause DNA damage in the isolated whole mammary gland and induce neoplastic transformation of the epithelial cells in a serum-free medium, in vitro.
Materials and Methods Animals
All mice used were of the BALB/c strain obtained from the National Institutes of Health (NIH) approved vendors. The BALB/c strain of mice does not express the endogenous murine mammary tumor virus and has a low occurrence of spontaneous mammary tumors (DeOme et al. 1980) [3]. Animal care was in accordance with standards set by the NIH. Organ culture
Mammary glands were cultured as previously described by Banerjee et al. (1983) [ 11. Briefly, 3-4-week-old BALB/c virgin female mice were primed by daily S.C. injections of progesterone (1 .O mg) and 17fi-estradiol (1.0 pg) from Sigma Chemicals for 9 days prior to culture. On the 10th day the mice were euthanized by cervical dislocation and the whole second thoracic mammary gland, including the primary duct and nipple, were aseptically excised on a Dacron raft. The mammary gland resting on the raft was transferred into a 60 x 15 mm plastic culture dish containing 4 ml of Waymouth’s MB752/2 culmedium (Grand Island Biological ture Company) supplemented with the following steroid and peptide hormones obtained from Sigma, St. Louis, MO; insulin, prolactin and hydrocortisone (5 pg/ml) and aldosterone (1 pg/ml). Penicillin was added at a concentration of 3.5 pg/ml. This medium is hereafter referred to as the mammogenic medium. The glands (4 glands to a culture dish) were placed in a 37OC humidified chamber with an atmosphere of 95% oxygen and 5% carbon dioxide. The media was changed every other day. Approximately 5% of the glands from each culture were stained and microscopically examined for lobulo-aveolar development. The staining procedure was as follows: glands were fixed for 1 h in modified Carnoy’s solution (ethanol and glacial acetic acid 3:l v/v), washed for 15 min in 70% ethanol, rinsed in distilled water, stained for 12 h in alum car-
33
mine, dehydrated in graded concentrations of alcohol and defatted in toluene for microscopic examination. Carcinogen treatment MNU was dissolved in dimethylsulfoxide (DMSO) at a concentration of 33 pg/pl and added to the cultures within 3 min of preparation. In the transplantation assay, the glands were treated twice with a MNU dose of 125 pg/ml medium. Both MNU treatments were each 45 min in duration and occurred once on day 3 and once on day 7 of the culture period. After the 7th day in culture, the glands were retained in the mammogenic media for 6 more days to ensure recovery from the carcinogen treatment. For the nick translation assay, separate cultures received a l-h MNU treatment of 250 pg/ml medium on different days of the mammogenic culture period. Tissue dissociation The tissue dissociation procedure was modified from that described by Richard et al. (1983) [12]. Briefly, MNU treated and DMSO treated (control) mammary glands were removed from the Dacron rafts using sterile forceps and blotted dry on a sterile Whatman qualitative filter. Thirty glands from each experimental group were pooled, placed in a sterile petri dish and weighed. The tissue was minced finely with crossed scalpels until the pieces were less than 1 mm3. The minced tissue was transferred into a trypsinization flask containing a sterile solution of collagenase in the ratio of 1 g tissue : 6 mg collagenase type III (Sigma) : 10 ml medium 199 (Grand Island Biological Company). The flask was shaken at 70 rev./min for 3 h at 37OC. The cell suspension was transferred to sterile centrifuge tubes and centrifuged at 180 X g for 10 min. The supernatant was discarded and the cell pelIet was resuspended and transferred to another flask containing 0.04% DNase and pronase in the ratio of 1 g original tissue : 1 mg pronase : 10 ml medium 199. The flask was shaken at 70 rev./min for 10 min at 37OC. The cell sus-
pension was poured through a sterile gauze filter to remove large cell clumps and fatty tissue. The cells were pelleted at 180 x g for 10 min and resuspended in 1 ml of medium 199. The cells were counted on a hemocytometer and the viability was determined using the Trypan blue exclusion test. Erythrocytes and white blood cells were not counted. The suspensions were discarded, if the cells in clumps could not be counted, or if the viability was less than 80%. Transplantation Three-week-old BALB/c virgin female mice were used as the recipients for the carcinogentreated cells. The host animals were anesthetized with nembutal solution (0.06 mg/g body wt.). Mice under 10 g were not used. The inguinal mammary glands were exposed by a midventral incision. Rudiments of the inguinal mammary gland including host epithelial tissue were removed by cauterization. Using a Hamilton No. 710SN syringe fitted with a 0.5-inch 30 gauge needle, 0.1 ml of the cell suspension (1 x lo5 cells) was slowly injected into the center of the remaining fat pad. In the left inguinal fat pad the MNU treated cells were injected and in the right inguinal fat pad the DMSO treated (control) cells were injected. The animals were then sutured and maintained in separate cages as virgins. The animals were palpated every week for the presence of tumors, and after 4 months all animals not bearing tumors were reopened and examined for the presence of mammary hyperplasia. Tissue thought to be hyperplastic was transplanted into syngeneic host animals to test its tumorigenic potential. This procedure involved placing a small piece (1 mm3) of the abnormal tissue into the cleared inguinal fat pad of a 3-week-old BALB/c virgin female mouse. In addition, small pieces of both hyperplastic and tumorous tissue were fixed in acetic alcohol, dehydrated in graded concentrations of alcohol, embedded in paraffin, sectioned at 5 PM and stained in hematoxylin and eosin for histological examination. After 10 weeks, all
34
mice secondarily transplanted with tissue originally thought to be hyperplastic or tumorous were reopened and examined for mammary outgrowths having the same morphology as the’primary transplanted tissue. Also, small pieces (1 mm3) of the tumorigenie tissue were subcutaneously transplanted onto the dorsal surface of syngeneic host BALB/c virgin female mice. Mice developing subcutaneous tumors were reopened and a small piece of the tumor was taken for histological examination. Nick translation The nick translation assay was based on the procedure developed by Nose and Okamato (1983) [ 111. Mammary cells previously isolated via the tissue dissociation procedure were resuspended in 5 ml of a solution containing 0.25 M sucrose, 0.1 M Tris-HCI (pH 7.4), 10 mM MgCI, and 0.5 mM dithiothreitol. LysoIecithin (100 pg/ml) was added just before use. The cells were kept on ice for 5 min then centrifuged at 180 x g for 10 min. The cell pellet was resuspended in the nick translation assay mixture containing 50 mM Tris-HCI (pH 7.4), 5 mM MgCI,, 10 mM 2-mercaptoethanol, 50 pg/ml bovine serum albumen, 0.05 mM of each dATP, dGTP and dTTP and 5 @/ml [3H]dCTP. E. coli DNA-polymerase I (50 units/ml) were then added and the reaction was allowed to proceed at room temperature for 30 min. After the reaction period, the entire mixture was applied to Whatman-3MM filter disks pre-wetted with 2% pyrophosphate on a Millipore suction filter. The filter disks were extensively washed with 5% trichloroacetic acid containing 1% pyrophosphate and
Table 1.
Neoplastic transformation
of mammary
rinsed with 100% ethanol. After drying, the acid insoluble radioactivity was measured by scintillation spectrometry on a Beckman 1201 liquid scintillation counter. The nick translation assays were conducted immediately after the 1 h carcinogen treatment. Results and Discussion To establish the neoplastic transformation potential of the direct-acting carcinogen on mammary epithelial cells in organ culture of the whole mammary gland in vitro, a transplantation assay was performed. The ability of MNU to induce neoplasm in the host mouse mammary gland was examined by injecting the cells from dissociated MNU treated glands vitro, into virgin female BALB/c host mice mammary fat pads, cleared of host epithelial tissue to prevent competition. The first set of transplantation experiments using cultured mammary glands treated with a single dose of MNU (250 pg/mI) failed to induce tumors or HANS in the glands of syngeneic host animals (Table I). Miyamoto et al. (1988) [9] had reported mammary cell transformation in a cell culture model using two treatments of MNU, so the tumorigenic potential of gland treated twice with MNU in culture was tested. Thirteen animals were used in the transplantation assay to test mammary glands treated twice in culture with MNU (125 pg/ml), once on day 3 and day 7. This carcinogen treatment gave rise to both tumors and HANS. Twentythree percent (3 mice) of the transplanted host mice developed palpable tumors within 3-4 months. An additional 31% (4 mice) of the transplanted host mice showed abnormal
epithelial cells in organ culture.
Experiment
Treatment
No. of fat pads
5%HANS
46 Tumors
I
1 MNU treatment 1 DMSO treatment 2 MNU treatments 2 DMSO treatments
42 42 13 13
0 0 31 0
0 0 23 0
I II II
All animals were successfully transplanted.
Fat pads not containing
HANS or tumors were normal in development.
35
hyperplastic growth. Pieces of all tumors and HANS were taken for histological examination and secondary transplantation. All hyperplasias were alveolar in origin with no ductal hyperplasias observed (Fig. 1). Tumor sections were determined to be adenocarcinoma of mammary epithelial cell origin. Figure 2 shows the invasive cellular extensions of the carcinoma. The secondary transplants all develabnormal oped outgrowths having a morphology and histology similar to their primary outgrowths within 10 weeks after transplantation. None of the parenchyma-free mammary fat pads of the host mice transplanted with cells from the dissociated DMSO treated (control) glands developed abnormal tissue. The final test for true adenocarcinoma is growth of the tumor at an ectopic site. Small
Fig. 2. Histological section gland showing the invasiveness induced try MNU.
Fig. 1. mammary lar origin.
Histological section of MNU treated mouse gland showing that the hyperplasia is of alveo-
of a mouse mammary of the adenocarcinoma
pieces of the tumorous tissue were S.C. transplanted onto the backs of 12 host BALB/c mice. Sixty-seven percent (8 mice) of the S.C. transplanted mice developed adenocarcinomas. Mouse mammary carcinogenesis in vivo is associated with the appearance of the HANS, followed by mammary tumors which appear later in the hyperplasia. Studies have shown that HAN tissue is serially transplantable into the parenchyma-free mammary fat pad of syngeneic mice. Prior to the tissue dissociation, MNU treated glands from each day of the 7 days mammogenie period, in culture, were placed separately in regression media (Waymouth’s MB752/2 culture medium supplemented with only insulin and penicillin) for 15 days and stained as previously described. Stained glands were assayed microscopically for the presence
36
of NLAL representing foci of cells which have escaped from normal hormonal control and remained unregressed indicating damage caused by the carcinogenic treatment (Fig. 3). Studies in our laboratory have demonstrated that the chemical carcinogen 7,12dimethylbenz[a]anthracene (DMBA) can induce neoplastic transformation of the mammary cells in a serum-free hormonally defined culture medium (Iyer and Banerjee, 1981) [6]. Additional studies have shown that the neoplastic transformation of the mammary gland in vitro is associated with the appearance of NLAL which, like HAN, are serially transplantable into the parenchyma free mammary fat pad of syngeneic female mice. Since the appearance of HAN and NLAL precedes the tumor formation, HAN and NLAL are designated as precancerous and HAN and NLAL
are related to neoplastic transformations. In this respect the neoplastic transformations induced by MNU and by DMBA appear to be rather similar and the studies described in this paper clearly indicate that the direct acting carcinogen MNU can induce neoplastic transformation of the mammary cells in organ culture. At present there is no report of a DNA damage study showing that the neoplastic transformation of the mammary cells induced by MNU can be caused by DNA damage in the mammary cells in vitro. Although an earlier study has reported that the direct acting carcinogen MNU can induce a high frequency of sister chromatid exchange (SCE). But the high frequency of SCE induced by MNU represents chromosomal rearrangements rather than DNA damage. This deficiency provided the rationale for including the quantitative measurement of DNA damage using the nick-translation assay. DNA damage was observed during all 6 days of the mammogenic period, with the greatest damage occurring between day 4 and day 5 of culture (Table II). Explants from control glands treated with DMSO consistently
Table II. MN&induced DNA damage as measured by nick-translation assay in mouse mammary epithelial cells during different stages of mammary development. Day treated
Treatment
Incorporation cpm/105 cells*
1 2
MNU DMSO MNU DMSO MNU DMSO MNU DMSO MNU DMSO
3840 610 6900 425 7204 349 7555 699 6035 561
3 3 4 4
Fig. 3. Wholemount of a stained mouse mammary gland treated with MNU showing the presence of nodulelike alveolar lesions (NLAL) Arrow indicates NLAL.
5 5 6 6
‘Each value is the mean of 3 separate different batches of glands.
f f & f f + * + f f
32 29 25 56 55 60 45 55 87 45
experiments
with
37
showed a basal level of DNA damage, as evidenced by a lower incorporation of [3H]deoxycytidylate. This low level of radioactivity indicates a basal level of DNA damage occurring within the cell. In cells treated with the carcinogen, the level of incorporation was at least lo-fold greater than in the control cells treated with DMSO. Consideration of the findings discussed in the preceding paragraphs reiterate the concept that components of the intact cells interact with the direct-acting carcinogens thus inducing the neoplastic transformation of the mammary epithelial cells. This observation is consistent with the possibility that MNU can functionally activate the expression of the endogenous oncocells. Recently, genes in the mammary Miyamoto et al. (1990) [lo] have indicated that mouse mammary epithelial cells transformed in primary culture with MNU have a specific mutation in the c-Ki-ras proto-oncogene and that this mutation is a preneoplastic event in mouse mammary carcinogenesis induced by MNU.
References 1
2 3
4 5
6
7
8
9
Acknowledgements We thank Dr. Sanjoy K. Das and Dr. Abhik M. Bandyopadhyay for their comments and discussion during preparation of the manuscript, Michelle Mathiesen for her technical assistance, Robert Schmaltz for editing assistance, Rodney S. Markin, M.D., Ph.D., Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, for identification of the pathological type of the tumors and Donna M. McCarthy for her secretarial assistance. This supported by Grant investigation was CA25304 awarded by the National Cancer Institutes. USPHS.
10
11
12
13
Banerjee, M.R. (1983) In: In Vitro Model for Cancer Research. Editors: M. Weber and L. Sekely. CRC Press, Boca Raton. Becker, F.F. ed. (1982) Cancer 1, a comprehensive treatise. 2nd edn., p. 263. Plenum Press, New York. DeOme, K.B., Osborne, R.C. and Miyamoto, M.J. (1980) In: Cell Biology of Breast Cancer, p. 79. Editors: C.C. McGrath, M. Brennan and M. Rich. Academic Press, New York. Grover, P.L. (1978) Chemical carcinogens and DNA. CRC Press, Boca Raton. Gullino, P. (1975) N-nitrosomethylurea as mammary gland carcinogen in rats. J. Natl. Cancer Inst., 54, 401411. lyer, A. and Banerjee, M. (1981) Sequential expression of preneoplastic and neoplastic characteristics of mouse mammary epithelial cells transformed in organ culture. J. Natl. Cancer Inst., 66,893-899. Lin, F.K., Banerjee, M.R. and Crump, R.L. (1976) Cell cycle-related hormone-carcinogen interaction during chemical carcinogen induction of NLAL in organ culture. Cancer Res., 36, 1607. Manoharan, K. and Banerjee, M.R. (1985) Measurements of chemical carcinogen-induced sister-chromatid exchanges in a whole organ in vitro. Mutat. Res., 147, 165- 169. Miyamoto, S., Guzman, R., Osborne, R. and Nandi, S. (1988) Neoplastic transformation of mouse mammary epithelial cells by in vitro exposure to N-methyl-N-nitrosourea. Proc. Natl. Acad. Sci. USA, 85,477-481. Miyamoto, S., Sukumar, S., Guzman, R.C., Osborn, R.C. and Nandi, S. (1990), Transforming c-Ki-ras mutation is a preneoplasttc event in mouse mammary carcinogenesis induced in vitro by N-methyl-N-nitrosourea. Mol. Cell. Biol., 10, 1593-1599. Nose, K. and Okamato, H. (1983) Detection of carcinogen-induced DNA damage by nick translation in permeable cells. Biochem. Biophys. Res. Commun., 111,383. Richard, J.D., Pasco, R., Guzman, R. and Nandi, S. (1983) Comparison for the growth of normal and neoplastic mouse mammary cells on plastic and collagen gels. Exp. Cell Res., 146, l-14. Sukumar, S., Notario, V., Martin-Zanca, D. and Barbacid, M. (1983) Induction of mammary carcinomas in rats by MNU involves malignant activation of H-ras-1 locus by single point mutations. Nature, 306,658-661.
