Transforming c-Ki-ras mutation is a preneoplastic event in mouse mammary carcinogenesis induced in vitro by N-methyl-N-nitrosourea.

Mouse mammary epithelial cells can be transformed in primary cultures to preneoplastic and neoplastic states when treated with N-methyl-N-nitrosourea (MNU). Mammary carcinomas arising from MNU-induced hyperplastic alveolar nodules (a type of mouse mammary preneoplastic lesion) contained transforming c-Ki-ras genes when examined by the NIH 3T3 focus assay. Hybridization of allele-specific oligonucleotides to c-Ki-ras sequences amplified by the polymerase chain reaction demonstrated the presence of a specific G-35----A-35 point mutation in codon 12 in each of the NIH 3T3 foci as well as the mammary carcinomas. This mutation resulted in the substitution of the normal glycine with an aspartic acid. Furthermore, this mutation in the c-Ki-ras proto-oncogenes was also detected in 9 of 10 hyperplastic alveolar nodules. These results demonstrate that the specific c-Ki-ras mutation is a preneoplastic event in MNU-induced mouse mammary carcinogenesis.

Effects of phorbol esters on normal and tumorous mouse mammary epithelial cells embedded in collagen gels.

The effects of phorbol esters on mammary epithelial cells from BALB/cfC3H/Crgl "midpregnant mice" (i.e., mice at the midterm of pregnancy) and from mammary adenocarcinomas (also from BALB/cfC3H/Crgl mice) grown in a collagen gel matrix were studied. 12-O-Tetradecanoylphorbol 13-acetate (TPA), when added to the media, caused an increased proliferation of both normal and cancerous mammary epithelial cells in a dose-dependent manner. The degree of enhancement of proliferation by TPA ranged from no increase in cell number at 3% swine serum (SW) concentration to two to three times the number of cells in the control cultures when 10 or 25% SW was used. Optimal growth was obtained with a TPA concentration of 0.1 or 1.0 microgram/ml. Increasing the SW concentration (3, 5, 10, or 25%) enhanced the proliferative effect of TPA. Cholera toxin (0.01 microgram/ml) enhanced the proliferative effect of TPA on normal cells but had a variable effect on tumor cells. The addition of TPA also resulted in a morphologic change in the epithelial colonies from midpregnant mice and from mammary tumors and caused them to assume a fibroblastic appearance. The addition of 4 alpha-phorbol or 4 alpha-phorbol 12,13-didecanoate to mammary epithelial cultures had no proliferative or morphologic effect. The results demonstrate that TPA has a growth-promoting effect on normal and cancerous mouse mammary epithelial cells.

Cultured mouse mammary epithelial cells: normal phenotype after implantation.

Mammary epithelial cells from normal virgin BALB/c mice were cultivated in vitro by the feeder cell technique developed and reported previously. These cells were cultured up to the 10th passage, equivalent to 60 cell divisions in culture. They were then tested for normality by several criteria, namely, the ability to regrow into normal mammary glands after implantation into cleared mammary fat pads of both syngeneic and nude mice, chromosome numbers, and response to mammogenic hormones. The cultured cells did form normal mammary ducts after implantation. The fraction of fat pads with ductal outgrowths as well as the size of the outgrowths was proportional to the number of cells implanted. When 10(6) cells were implanted into BALB/c mice, 83% of the fat pads contained outgrowths, filling, on the average, approximately 87% of the fat pad. More ductal outgrowths occurred from implanted cells taken from lower tissue culture passages than from high ones, and the number of outgrowths was greater in BALB/c mice than in nude mice. A small fraction of the cells in culture reacted with antibodies to casein, but there was no evidence of alpha-lactalbumin in the cells. However, ductal outgrowths from implanted cells responded to hormone stimulation of an estrogen deoxycorticosteroid pellet by forming well-developed lobulo-alveolar structures characteristic of pregnancy. Of the cells that were studied in passages 3 and 7, 85% were diploid. An abnormally growing culture in passage 10 was composed of cells in the tetraploid range. These tetraploid cells formed normal mammary ducts when implanted into animals.

Recovery of transformed nodule and ductal mammary cells from carcinogen-treated C57BL mice.

Transformed nodule and ductal mammary cells were recovered by cell dissociation and transplantation of mammary cells from C57BL/Crgl mice treated with 7,12-dimethylbenz(a)anthracene. Four-week-old mice were divided into the following groups: Group A, not treated; Group B, 2 pituitary isografts under the kidney capsule; Group C, 1.0 mg 7,12-dimethylbenz(a)anthracene intragastrically at 5 and 6 weeks of age; and Group D, 2 pituitary isografts and 1.0 mg 7,12-dimethylbenz(a)anthracene intragastrically at 5 and 6 weeks of age. At 10, 14, 18, and 22 weeks of age, the mammary glands were enzymatically dissociated, and 10(5) cells were injected into the gland-free mammary fat pads of 3-week-old syngeneic mice. After 10 weeks, the outgrowths were examined and classified as ductal, ductal dysplasia, or hyperplastic alveolar nodule. Ductal dysplasias and hyperplastic alveolar nodule outgrowths were recovered from carcinogen-treated mice. Pituitary isografting enhanced the recovery of ductal dysplasia. Five serially transplanted dysplastic outgrowth lines were established and are their fifty and sixth transplant generations. The data demonstrate that transformed mammary gland cells can be removed from carcinogen-treated mice by means of cell dissociation and transplantation.

Transfection of activated Ha-ras protooncogenes causes mouse mammary hyperplasia.

ras protooncogenes activated by a point mutation have been implicated in the initiation of mammary carcinogenesis. However, the nature of phenotypic alterations induced by activated ras protoonocogenes during initiation has not been well understood. In the present studies, the phenotypic manifestation of activated ras genes was directly analyzed by transfecting them into normal mouse mammary epithelial cells. The ras genes were cotransfected with pSV2neo which expresses the bacterial neomycin resistance gene to partially select for successful transfectants in culture. Transfection of activated Ha-ras protooncogenes, containing a point mutation in codon 12, caused hyperplasia in the mouse mammary gland following transplantation. Hyperplastic phenotype is a prerequisite for neoplastic development. The hyperplasias induced by the activated Ha-ras protooncogenes, however, were not immortal in vivo, another essential characteristic of preneoplastic and neoplastic mouse mammary cells. Control cells transfected only with pSV2neo did not produce any hyperplasia. These results suggest that a functional role of activated ras protooncogenes in the initiation of mouse mammary carcinogenesis may be the induction of a hyperplastic phenotype, a prelude to neoplastic development.

In vitro transformation of mouse mammary epithelial cells grown serum-free inside collagen gels.

Mammary epithelial cells from 4-month-old virgin BALB/c mice were cultured inside collagen gels in the following serum-free media: Dulbecco's modified Eagle's medium:Hams's F-12 (1:1) supplemented with: insulin (10 micrograms/ml), bovine serum albumin (5 mg/ml), and epidermal growth factor (5 ng/ml); insulin, bovine serum albumin, progesterone (0.05 microgram/ml), and prolactin (1 microgram/ml); insulin, bovine serum albumin, progesterone, prolactin, and linoleic acid (10 micrograms/ml). Cells proliferated in all these media. The cells were treated with 0.01 micrograms/ml of 7,12-dimethylbenz(a)anthracene or 100 micrograms/ml of N-nitroso-N-methylurea on day 3 of culture and, subsequently, at 1-week intervals for 3-6 weeks. Tetradecanoylphorbol acetate (0.1 micrograms/ml) was added to selected cultures. The cultures were maintained for up to 9 weeks; the cells were then removed from the collagen gels, placed in monolayer culture for 2 days, and removed from monolayer culture, and 5 X 10(5) cells were transplanted to each of the gland-free mammary fat pads of 3-week-old female mice. Approximately 10 weeks after transplantation, the transplanted mammary fat pads were examined for outgrowths. Cells that were not treated with carcinogen and cultured for up to 9 weeks in different serum-free media and transplanted to the gland-free mammary fat pad produced only ductal outgrowths similar in morphology to the ducts of the virgin host's mammary glands. Six treatments with 7,12-dimethylbenz(a)anthracene, of cells grown in the presence of epidermal growth factor, induced 31% spindle cell tumors, 17% ductal hyperplasias, and 5% lobuloalveolar hyperplasias. Cells that were grown in epidermal growth factor and treated three times with N-nitroso-N-methylurea produced 23% ductal hyperplasias and 17% lobuloalveolar hyperplasias. Cells grown in the presence of progesterone and prolactin and treated three times with 7,12-dimethylbenz(a)anthracene produced up to 23% lobuloalveolar hyperplasias and 12% ductal hyperplasias. Three treatments with N-nitroso-N-methylurea of cells grown in progesterone- and prolactin-containing media produced a maximum of 50% lobuloalveolar hyperplasias and 33% ductal hyperplasias. The lobuloalveolar hyperplasias have the characteristics of the precancerous hyperplastic alveolar nodules found in mouse mammary tumorigenesis. The in vitro carcinogen-induced lobuloalveolar hyperplasias were transplantable, maintained their lobuloalveolar morphology in virgin hosts, and produced carcinomas.

Role of endocrine, autocrine, and paracrine interactions in the development of mammary hyperplasia in Wnt-1 transgenic mice.

The Wnt-1 proto-oncogene is transcriptionally activated by mouse mammary tumor virus in mouse mammary tumor virus-induced tumors. Previous studies using transgenic mice showed that Wnt-1 expression in mammary gland causes alveolar hyperplasias which resemble mammary glands of pregnant mice. To understand the role of mammogenic hormones in the genesis of these hyperplasias, we examined the development of these glands before puberty in young transgenic mice and the effects of ovariectomy and adrenalectomy on the growth and morphology of Wnt-1 mammary hyperplasia. Mammary glands of Wnt-1 transgenic females showed hyperplastic morphology as early as 1 week after birth. The normal structure of the uterus of the adult Wnt-1 virgin mouse indicated that the circulating levels of ovarian hormones were not elevated. Ovariectomy and adrenalectomy had no obvious effect on the morphology of these mammary hyperplasias. To assess possible paracrine stimulation of mammary epithelial cells (MEC) by stromal cells, we transplanted MEC from normal BALB/c mice into gland-free fat pads of Wnt-1 transgenic mice and found that normal MEC maintained their normal ductal structure in Wnt-1 fat pads without alveolar development. Further, we did not detect Wnt-1 mRNA expression in the gland-free fat pads of these transgenic mice. When Wnt-1 MEC were transplanted into the fat pads of nude mice and allowed to grow towards existing normal MEC, the morphology of the existing normal MEC remained normal. We concluded that the development of mammary hyperplasia in Wnt-1 transgenic mice is solely dependent on Wnt-1 expression in MEC. We speculate that Wnt-1 may be a growth factor for mammary gland that only acts locally on the cells that produce it.

Transplantation of mouse mammary epithelial cells grown in primary collagen gel cultures.

A technique for the transplantation of mouse mammary epithelial cells, grown in collagen gels, has been developed that demonstrates that the phenotype of the cells prior to culture was not altered by the culture conditions. Mammary epithelial cells from virgin and midpregnant C57BL/Crgl mice; virgin, midpregnant, and multiparous nonpregnant BALB/cfC3H/Crgl mice; a BALB/c hyperplastic alveolar nodule line, and mammary tumors from BALB/cfC3H/Crgl mice were embedded inside collagen gels and grown for 10 to 14 days in the presence of 25% swine serum plus cholera toxin (0.01 microgram/ml). The epithelial cells increased in number during the culture period. At the end of the culture period, the cells were removed from the collagen gels and transplanted to the gland-free mammary fat pads of 3-week-old syngeneic female mice. Culture in collagen gels increased the number of cells necessary to obtain a high percentage of mammary outgrowths as compared to cells not grown inside collagen gels. In general, mammary cells grown inside collagen gels gave rise to outgrowths, similar in phenotype to those from noncultured cells, and were representative of the tissue of origin. Mammary epithelial cells from C57BL/Crgl virgin donors grown in collagen gels for 10 to 14 days retained their ability to respond to the endogenous hormones of pregnancy and lactation of the host and formed lobuloalveolar structures full of secretion similar to the host's own mammary gland. The data indicate that the growth of mammary epithelial cells in collagen gels and subsequent transplantation into the gland-free fat pads of syngeneic hosts provides a simple system, wherein cells can be grown in vitro and their phenotypes determined in vivo.

Effect of parity on recovery of inapparent nodule-transformed mammary gland cells in vivo.

Inapparent nodule-transformed cells were recovered from five late-pregnant, first-pregnancy BALB/cfC3H females 4 months of age and from five late-pregnant multiparous females 6 to 7 months of age. Mammary tissues were removed from each donor and dissociated by means of the enzyme collagenase (0.1%), hyaluronidase (0.1%), and pronase (1.25%). Aliquots of 100,000 viable cells in 0.01 ml of media were injected into the gland-free mammary fat pads of 3-week-old syngeneic host mice. Ten weeks after the injection the outgrowths were classified as ductal, nodule, tumor, or combinations of these types of outgrowths. The recovery of nodule outgrowths indicated the presence of nodule-transformed cells in the cell suspension that was injected. All donors yielded nodule outgrowths, and the percentage of outgrowths was significantly greater than was the percentage recovered from virgin BALB/cfC3H females of the same age groups. The latent period for the emergence of nodules and tumors was reduced from 8 to 9 months in virgin females to 4 months in parous females. The incidence of both nodules and tumors was greatly increased. The data suggest that parity significantly increases the numbers of nodule-transformed cells in donor tissue, decreases the time required for the emergence of nodules and tumors, and increases the number of overt nodules and tumors.

Detection of inapparent nodule-transformed cells in the mammary gland tissues of virgin female BALB/cfC3H mice.

Inapparent nodule-transformed cells were recovered from morphologically normal mammary tissue from virgin female BALB/cfC3H/Crgl (mouse mammary tumor-positive) mice before the appearance of hyperplastic alveolar nodules (HAN) or tumors, by means of the cell dissociation technique. Mammary tissues were dissociated by means of collagenase (0.1%), hyaluronidase (0.1%), and pronase (1.25%). Aliquots of 10(5) viable cells in 0.01 ml medium were injected into the gland-free mammary fat pads of 3-week-old female syngeneic mice. After 10 weeks the outgrowths were examined and classified as ductal, HAN, tumor, or combinations of these types. The presence of HAN outgrowths indicated the presence of nodule-transformed cells in the donor mammary tissues. Nodule-transformed cells were recovered from 2-month-old donors, and the number of HAN outgrowths increased with donor age. Overt HAN and tumors did not appear in virgin female BALB/cfC3H/Crgl mice younger than 8 to 9 months of age. The data suggest that inapparent nodule-transformed cells occurred long before the appearance of HAN of tumors and that the numbers of transformed cells increased with donor age.

Incidence of c-Ki-ras activation in N-methyl-N-nitrosourea-induced mammary carcinomas in pituitary-isografted mice.

We found previously that mouse mammary epithelial cells cultured in the presence of the mammogenic hormones progesterone and prolactin and treated with the carcinogen N-methyl-N-nitrosourea produced a high frequency of hyperplastic alveolar nodules and carcinomas with squamous metaplasia upon transplantation to syngeneic mice. The majority of these mammary transformants had an activated c-Ki-ras proto-oncogene with a specific point mutation in codon 12 (G35 to A35). To determine whether these in vitro findings parallel mammary carcinogenesis in vivo, virgin female mice were pituitary isografted to increase their circulating levels of progesterone and prolactin. The pituitary isograft results in an increase in proliferation, leading to lobulo-alveolar development and differentiation of the mammary epithelial cells. Five weeks after pituitary isografting, the mice were treated with a single injection of N-methyl-N-nitrosourea (50 micrograms/g body weight). Greater than 90% of the N-methyl-N-nitrosourea-treated mice developed mammary carcinomas between 3 and 7 months after treatment. The majority (75%) of the carcinomas had histopathology identical to that of tumors induced in vitro in the presence of progesterone and prolactin. A number of the mammary cancers (17%) induced in pituitary-isografted mice also had the identical point mutation in the c-Ki-ras proto-oncogene found in the in vitro studies. These results suggest that the hormonal milieu around the time of carcinogen exposure affects not only the incidence and phenotype of the mammary transformants but also the molecular events associated with mammary carcinogenesis.

Overexpression of transforming growth factor alpha overrides the glucocorticoid-mediated suppression of Con8 mammary tumor cell growth in vitro and in vivo.

In a preceding paper (D. B. Alexander et al., Cancer Res., 53: 1808-1815, 1993), we demonstrated that the in vitro glucocorticoid inhibition of Con8 mammary tumor cell growth is accompanied by the disruption of a transforming growth factor alpha (TGF-alpha) autocrine loop. This growth suppression response functions in vivo since proliferation of Con8-derived tumors was inhibited in rats treated with the synthetic glucocorticoid, dexamethasone. The effect of dexamethasone on Con8-derived tumor growth was reversible in that tumors rapidly grew at the site of inoculation after discontinuing injections of dexamethasone. To test the in vivo relationship between the glucocorticoid growth suppression response and the TGF-alpha autocrine loop, Con8 cells were transfected with a TGF-alpha expression vector and single cell-derived neomycin-resistant subclones were recovered. [3H]Thymidine incorporation of cultured monolayers of transfected Con8 mammary cells and measurement of tumor diameters in rats revealed that dexamethasone failed to suppress the in vitro proliferation or in vivo tumor growth of Con8-derived cells producing high constitutive levels of secreted TGF-alpha. In contrast, both the in vivo and in vitro growth of Con8 cells transfected with vector controls were fully suppressible by glucocorticoids. Consistent with our in vitro observations, these results demonstrate that the regulation of TGF-alpha production plays a key role in the in vivo glucocorticoid suppression of Con8-derived mammary tumor growth.

The 16-kilodalton N-terminal fragment of human prolactin is a potent inhibitor of angiogenesis.

The formation of a new blood supply, angiogenesis, is an essential component of carcinogenesis and unrestricted tumor growth. A substance capable of inhibiting angiogenesis would be of considerable therapeutic potential in the treatment of cancer. We previously reported that the 16-kilodalton N-terminal fragment of rat PRL (16K rPRL) was a potent inhibitor of capillary endothelial cell proliferation via a novel receptor. We now report that the nanomolar concentrations of recombinant human 16K PRL inhibit basal and basic fibroblast growth factor- or vascular endothelial growth factor-stimulated growth of bovine brain capillary endothelial cells. 16K human (h) PRL also inhibits stimulation of human umbilical vein endothelial cell proliferation by basic fibroblast growth factor. The organization of endothelial cells into capillary-like structures in type I collagen gels is also prevented by 16K hPRL. Furthermore, in an in vivo assay, the chick embryo chorioallantoic membrane assay, 16K hPRL as well as 16K rPRL were potent inhibitors of capillary formation. 16K hPRL, like 16K rPRL, maintains its biological activity as a partial PRL agonist at PRL receptors on mammary gland epithelial cells. These data demonstrate for the first time that the biological activity of 16K rPRL is not unique and that similar fragments of hPRL are active. The antiangiogenic activity of these molecules is conserved across avian and mammalian species. That 16K hPRL is a potent antiangiogenic factor in in vitro and an in vivo assay raises the exciting potential of this peptide being capable of inhibiting tumor growth.

An in vitro model of epithelial cell growth stimulation in the rodent mammary gland.

Mouse mammary epithelial cell cultures previously described bring about extensive proliferation and a cell population with the appropriate markers for luminal ductal epithelial cells, and also the ability to form normal tissue after implantation into mice. This success may result from a culture environment that resembles certain aspects of the environment in the mammary gland. Mouse mammary epithelial cells, whose proliferation is limited when plated alone, can be stimulated to multiply by contact with lethally irradiated cells of the LA7 rat mammary tumour line. Most of the proliferative stimulus is imparted by direct cell contact between LA7 and mouse mammary cells. Junctions, including adherens junctions, form among all cells in the culture, much as junctions form in the mammary gland. LA7 cells secrete TGFalpha and bFGF, factors found in the mammary gland, and factors to which mouse mammary cells respond in culture. Mouse mammary cells express keratins 8 and 18, markers for luminal cells of the mammary duct. LA7 cells express keratin 14 and vimentin, markers for myoepithelial cells. These facts, taken together, fit a model of cell replacement in an epithelial tissue and also imitate the relationship between luminal ductal cells and myoepithelial cells in the mammary gland. This method of culturing cells is useful, not only for in vitro-in vivo carcinogenesis studies, but also for the study of mechanisms by which growth signals are imparted from one cell to another.

Metabolism of 7,12-dimethylbenz[a]anthracene by mouse mammary epithelial cells cultured serum-free inside collagen gels.

Mammary epithelial cells from 3-4-month-old BALB/c virgin mice were cultured inside collagen gels in the following serum-free media: Dulbecco's Modified Eagle's Medium/Ham's F-12 (1:1) supplemented with (A) insulin, bovine serum albumin, epidermal growth factor; (B) insulin, bovine serum albumin, progesterone, prolactin; (C) insulin, bovine serum albumin, progesterone, prolactin, linoleic acid. Cell number increased with all media used. At day 7 of culture, [3H]dimethylbenz[a]anthracene (DMBA) was added to the cultures and its metabolism to water soluble and organic soluble compounds was determined. Mouse mammary epithelial cells were able to metabolize [3H]DMBA to water and organosoluble metabolites. By 72 h, 77-94% of the added DMBA had been metabolized by the epithelial cells in the three media to water and organosoluble metabolites in equivalent amounts. The distribution between water soluble and organosoluble metabolites was approximately equivalent. The high pressure liquid chromatography profiles of organosoluble fractions from the media indicated that the major products appeared to be the phenols, 2-,3-, or 4-hydroxydimethylbenz[a]anthracene, the hydroxymethyl derivatives, 7-methylbenz[a]anthracene and 7-hydroxymethylbenz[a]anthracene, trans-3,4-dihydro-3,4-dihydroxydimethylbenz[a]anthracene and one or two major fractions eluting just behind the marker cis-5,6-dihydro-5,6-dihydroxydimethylbenz[a]anthracene. The major fraction eluting just ahead of the cis-5,6-dihydro-5,6-dihydroxydimethylbenz[a]anthracene was most likely trans-8,9-dihydro 8,9-dihydroxydimethylbenz[a]anthracene. The profiles were similar for the cells cultured in all three serum-free media. The results demonstrate that mouse mammary epithelial cells cultured inside collagen gels with serum-free media can metabolize DMBA to putative carcinogenic forms.

Hormone dependent and independent mammary tumor development form N-methyl-N-nitrosourea-treated rat mammary epithelial cell xenografts in the nude mouse: multiple pathways and H-ras activation.

A nude mouse mammary fat pad xenograft system was developed to examine hormone dependent and independent mammary tumorigenesis and progression from N-methyl-N-nitrosourea (MNU)-induced hyperplastic lesions. Ninety-one percent of transplanted mammary tumors grew, with an orthotopic preference, and maintained their hormone dependence, histopathology, and H-ras mutation frequency. Grafted mammary epithelial cells, from MNU-treated rats, developed normal; and hyperplastic outgrowths, representative of those found in the rat mammary gland after MNU-treatment. Hyperplasias developed into neoplasias that were both hormone dependent and independent. We demonstrate that hormone independent tumors can develop directly either from lobuloalveolar or ductal hyperplasias or from hormone dependent tumors. H-ras mutation was detected in mammary preneoplasias (4 lines) before they developed into tumors and was associated with an elevated tumorigenic potential. Our observations suggest that there are multiple histopathogenic pathways in the development and progression to hormone independent rat mammary tumors.

Phenotypic characterization of collagen gel embedded primary human breast epithelial cells in athymic nude mice.

We have developed a method to characterize the phenotypes and tumorigenicity of dissociated human breast epithelial cells. The dissociated cells were first embedded in collagen gels and subsequently transplanted subcutaneously in vivo in athymic nude mice. The transplantation of dissociated epithelial cells from reduction mammoplasties, presumed to be normal, always resulted in normal histomorphology. Epithelial cells were arranged as short tubular structures consisting of lumina surrounded by epithelial cells with an occasional more complex branching structure. These outgrowths were surrounded by intact basement membrane and were embedded in collagen gel that, at termination, contained collagenous stroma with fibroblasts and blood vessels. In contrast, transplantation of dissociated breast epithelial cells from breast cancer specimens resulted in outgrowths with an invasive pattern infiltrating the collagen gel as well as frank invasion into vascular space, nerves and muscles. These observations were made long before the subsequent palpable stage which resulted if left in the mouse for a long enough time. The dissociated human breast epithelial cells thus retained their intrinsic property to undergo morphogenesis to reflect their original phenotype when placed in a suitable environment, the collagen gel.

Refractoriness to mammary carcinogenesis in the parous mouse is reversible by hormonal stimulation induced by pituitary isografts.

We have previously reported that mouse mammary epithelial cells transformed in vitro yield tumors which vary qualitatively and quantitatively as a function of the mitogenic environment in which the cells are propagated at the time of carcinogen treatment. One milieu supportive of transformation in vitro was medium supplemented with progesterone and prolactin as the mitogens. We have performed parallel studies in which virgin mice were isografted with pituitaries resulting in elevated serum titers of progesterone and prolactin. After carcinogen treatment, these mice developed mammary tumors which included those identical genotypically and phenotypically to tumors induced in vitro in cells grown in progesterone and prolactin during carcinogen exposure. Our current working hypothesis is that the mitogenic environment around the time of carcinogen administration can modulate the incidence and phenotype of the resultant tumors. To further test this hypothesis, we have evaluated the susceptibility of hormonally-stimulated parous mice to chemically induced mammary carcinogenesis since parity is known to significantly reduce the susceptibility of the mouse mammary gland to carcinogenesis. Virgin or multiparous BALB/c mice were isografted with two pituitaries. Five weeks after surgery, the mice were injected with N-methyl-N-nitrosourea (MNU; 50 micrograms/g i.v.). Mammary carcinomas arose in 85% (11/13) with a median latency of 22.8 weeks and 1.9 tumors per virgin mouse and 80% (24/30) with a median latency of 22.1 weeks at a frequency of 1.9 tumors per parous mouse. Only 14% (2/14) of the non-isografted, age-matched parous controls developed tumors when injected with MNU. Fourteen parous mice receiving only pituitary isografts (no MNU), did not develop mammary carcinomas within the 7-month period of the study. These results demonstrate that parous BALB/c mice are refractory to MNU-induced mammary carcinogenesis and that this refractoriness is not permanent, but can be overcome by hormonal stimulation mediated by pituitary isografts.

N-Ethyl-N-nitrosourea induces mammary cancers in the pituitary-isografted mouse which are histologically and genotypically distinct from those induced by N-methyl-N-nitrosourea.

N-Ethyl-N-nitrosourea (ENU) and N-methyl-N-nitrosourea (MNU) are alkylating agents which respectively ethylate or methylate nucleophilic centers in the cell such as DNA. In vitro studies with naked DNA and bacterial mutagenesis assays suggest that these two compounds induce different spectra of genetic lesions. In addition, the ethyl-DNA adducts induced by ENU persist longer than the methyl-DNA adducts induced by MNU. Since MNU is a known mammary carcinogen in the pituitary-isografted mouse, these data suggest that ENU may be an even more potent carcinogen than MNU. The purpose of this study was to determine whether ENU was a mammary carcinogen in the pituitary-isografted mouse and if so, to compare the genotype and phenotype of ENU-induced mammary tumors with those induced by MNU. Fifteen adult female virgin BALB/c mice were isografted with two pituitaries and subsequently treated with a single intravenous injection of ENU (50 micrograms/g body weight). Mammary adenocarcinomas arose in all of the survivors (n=12) with a median latency of 27 weeks and a mean frequency of 1.4 cancers per mouse. When tumor DNA was analyzed for mutations in the 12th and 61st codons of c-Ki-ras or c-Ha-ras protooncogenes, only wild type sequences were found. This is in contrast to MNU which causes a G to A transition mutation in the 12th codon of the c-Ha-ras proto-oncogene in about one of five mammary cancers induced in pituitary-isografted mice. Furthermore, the ENU-induced tumors were solid viable papillary adenocarcinomas, whereas MNU induced tumors are highly necrotic adenocarcinomas with squamous metaplasia. These results demonstrate that, in the pituitary-isografted mouse, ENU is as potent a mammary carcinogen as MNU and suggest that oncogenes other than c-Ki-ras or c-Ha-ras may be involved in ENU-induced mammary cancers.

Adenoviral-mediated gene transfer into primary human and mouse mammary epithelial cells in vitro and in vivo.

Primary mammary epithelial cells from both the human and mouse mammary glands can be genetically altered under a variety of situations using the replication-defective adenoviral vector containing a marker gene encoding the E. coli beta-galactosidase. Primary human and mouse mammary epithelial cells in monolayer culture and in three-dimensional collagen gel culture systems were transduced by adenovector at high efficiency. Successful gene transfer was also accomplished in situ and in vivo. In the mouse mammary gland, anatomically restricted gene transfer and expression was demonstrated by micro-injection of adenoviral vector directly into the main duct of the mammary gland. Injection of adenoviral vector directly into the human mammary tissues from reduction mammoplasty specimens, into the mouse mammary gland-free fat pad containing the previously transplanted dissociated human mammary epithelial cells, and intratumorally into the human breast cancer xenografts in nude mice, all resulted in successful gene transfer to human mammary epithelial cells. High efficiency introduction of genetic material into primary mammary epithelial cells is important in the study of mammary carcinogenesis and potentially for gene therapy of human breast cancer.