Hyperplastic Alveolar Nodules Research Articles

Immunoendocrine mechanisms in mammary tumor progression: direct prolactin modulation of peripheral and preneoplastic hyperplastic-alveolar-nodule- infiltrating lymphocytes.

We have previously shown that the immunoregulatory function of prolactin may play a role in the progression of the mouse mammary preneoplastic hyperplastic alveolar nodule (HAN) line C4 to carcinoma. In this study we investigated the direct effect of prolactin on lymphocytes isolated from normal and C4-HAN-bearing mice. In addition, we tested the effect of ovariectomy on prolactin/lymphocyte interaction to see whether, as has been reported in rats [Mukherjee P., Hymer W. C. (1992) Prog Neuroendocrinol Immunol 5: 108; Viselli S. M. et al. (1991) Endocrinology 129: 983], removal of estrogen would enhance the response to prolactin in mice. Proliferation of splenocytes, lymph node cells and HAN-infiltrating lymphocytes was stimulated by prolactin in a dose-responsive fashion. Ovariectomy did not alter this effect consistently. Cell-cycle analysis based on simultaneous staining of DNA and RNA revealed that prolactin-stimulated lymphocytes progress through all phases of the cell cycle whereas anti-prolactin antiserum inhibits this stimulation. Two-color flow-cytometric analysis revealed the time-dependent induction of interleukin-2 (IL-2) receptor expression on both CD4+ and CD8+ cells by prolactin. Prolactin-treated lymphocytes also produced low yet detectable levels of bioactive IL-2 in a dose- and time-dependent fashion. Prolactin enhanced lymphocyte responsiveness to mitogens and showed a marked synergism at suboptimal concentrations. Pretreatment of splenocytes from HAN bearers with a high concentration of prolactin slightly enhanced natural killer (NK) activity; anti-prolactin antiserum reduced the NK lytic activity of poly(I).poly(C)-activated splenocytes from HAN-bearing mice. Our results provide direct experimental evidence for the stimulatory effect of prolactin on lymphocyte function and IL-2-mediated lymphocyte proliferation and suggest a mechanism linking the endocrine system to immunomediated enhancement of HAN progression.

Infrequent p53 mutations in 7,12‐dimethylbenz[a]anthracene–induced mammary tumors in BALB/c and p53 hemizygous mice

We conducted experiments to determine if p53 alterations, which are frequent in human breast cancers, were also common in murine mammary tumors. In 13 mammary tumors from 7,12-dimethylbenz[a]anthracene (DMBA)-treated BALB/c mice were immunohistochemically analyzed for overexpression of p53; p53 protein was not detectable. Three of the tumors were established as cell lines in vitro. p53 protein was rarely detected at passage 4 in these lines but was overexpressed by passage 8 in two of them. The p53 nucleotide sequence was shown to be wild type in one primary mammary tumor and in the two p53-overexpressing cell lines. One cell line that overexpressed p53 in vitro was implanted into BALB/c mice. The resulting tumors retained the wild-type p53 nucleotide sequence but no longer expressed detectable levels of p53 protein, suggesting that the overexpression of wild-type p53 was related to in vitro culture conditions. The effect of DMBA on mammary-tumor development was also tested in mice rendered hemizygous for p53. These mice and wild-type littermate controls had no differences in susceptibility to induction of mammary tumors by oral administration of DMBA. Furthermore, Southern blot hybridization detected no gross alterations in the wild-type p53 allele in mammary tumors from the p53-deficient mice. Point mutation of the wild-type p53 allele was also infrequent in the DMBA-induced mammary tumors from hemizygous p53 mice; it occurred in only one of seven tumors. Thus, the p53 gene is apparently not a primary target for genetic alterations in DMBA-induced mammary tumors. Next, we examined mammary tumors derived from D1 and D2 transplantable hyperplastic alveolar nodule (HAN) outgrowths, which rapidly form tumors containing Ha-ras mutations after DMBA treatment. As ras and p53 mutants can cooperate in transformation, we examined whether D1 and D2 HAN outgrowths have p53 mutations. Unlike in the DMBA-induced primary mammary tumors, nuclear p53 accumulation was observed frequently (10 of 14) in tumors that arose from D1 and D2 HAN outgrowths. Direct sequencing of the entire coding region of the p53 cDNA from six D1 and D2 tumors confirmed that the sequence was wild type. Although wild-type p53 was retained in both DMBA-induced mammary tumors and mammary tumors derived from D1 and D2 preneoplastic outgrowths, wild-type p53 overexpression was detected only in D1 and D2 tumors. Therefore, D1 and D2 tumors appear to arise by a pathway in which p53 expression is altered, whereas DMBA induction affects a different pathway that does not require such alteration.

SUPPRESSION OF TUMORIGENESIS AND TUMOR-GROWTH OF MOUSE MAMMARY-GLANDS BY A TRADITIONAL CHINESE HERBAL MEDICINE, SHO-SAIKO-TO

Hyperplastic alveolar nodule (HAN) is a representative preneoplastic state in mammary glands of mice. We investigated the effect of Sho-saiko-to (SST; a Japanese modified traditional Chinese herbal medicine containing seven medicinal plants) on the formation and growth of HAN and mammary tumour growth in SHN virgin mice which showed a high incidence of spontaneous mammary tumours. Chronic oral administration of SST reduced the number and the area of HAN and mammary thymidylate synthetase (TS) activity with slight decrease of serum prolactin level. In the established mammary tumours of mice, 20-day administration of SST reduced the activity of TS and thymidine kinase (TK) with slight reduction of serum prolactin level, though the significant change of mammary-tumour growth was not observed. These results indicate that SST may prevent the mammary tumourigenesis by suppression of the de novo pathway for pyrimidine nucleotide synthesis and the mammary tumour growth by suppression of the salvage pathway as well as the de novo pathway in mice.

A new infectious mammary tumor virus in the milk of mice implanted with C4 hyperplastic alveolar nodules

We have previously characterized an infectious mouse mammary tumor virus [(MMTV(SW)] which induces a strong superantigen response in vivo. Here we describe the isolation and characterization of MMTV(C4) which was derived from milk of mice implanted with hyperplastic alveolar nodules. MMTV(C4) stimulates V beta 2 expressing T cells after local injection in vivo. Comparison with known open reading frame (orf) sequences revealed high homology to Mtv-6, an endogenous virus interacting with V beta 3-expressing T cells. The carboxyl-terminal amino acids were, however, altered. High homology including the carboxyl-terminal orf amino acids were found with MMTV(C3H-K). We show here that MMTV(C3H-K) has lost its superantigen function. Sequence comparisons permitted the characterization of few key amino acids which could be important for T cell receptor interaction and superantigen processing.

Suppression by kampo medicines in preneoplastic mammary hyperplastic alveolar nodules of SHN virgin mice.

Sho-saiko-to (SST), Keishi-bukuryo-gan (KBG), and Shakuyaku-kanzo-to (SKT) are Japanese modified traditional Chinese herbal medicines (Kampo medicines) consisting of 7, 5, and 2 medical plants, respectively. It is known that the hyperplastic alveolar nodule (HAN) is a representative preneoplastic state in the mammary glands of mice. We examined the effects of SST, KBG, and SKT on the formation and growth of HAN in a high-mammary-tumor strain of SHN virgin mice. Oral administration of SST for 60 days beginning at 90 days of age reduced the number and area of HAN and mammary thymidylate synthetase activity with a reduction of serum prolactin level. There was little difference between the other experimental groups and the control in the formation and growth of HAN and the enzyme activities. These results indicate that SST may have a preventive effect on malignant mammary transformations.

Growth of the mammary gland in rhino mouse.

The rhino mouse, so called because of its characteristic rhinocerous-like skin cannot raise its young. Although the animal has rudimentary mammary glands, these are thought to be defective due to their impossibility to raise their young. Morphological observations of the female mammary glands of rhino mice (hrrh/hrrh) were carried out and the results were compared with those of their normal litter mates (hrrh/+) at various ages from 1 month to 1 year. No morphological differences in mammary gland development was seen between the rhino mice and their normal litter mates, and normal secretory changes were observed in the rhino mice after delivery. Mouse mammary tumor virus (MMTV) expression and the occurrence of hyperplastic alveolar nodule (HAN) were analogous to these features in phenotypically normal hrrh/+ mice. Further, the sweat glands in the rhino mouse were normal. Thus, anomalies in the rhino mouse appeared to be limited to the skin and pilosebaceous apparatus.

The similar mammary tumour potentials in virgins and breeders of SHN mice.

Spontaneous mammary tumour potential was compared between virgins and breeders of a high mammary tumour strain of SHN mice. Virgins were essentially similar to breeders in both the cumulative and the monthly incidences of mammary tumours. DNA synthesizing enzyme activities and the number and the weight of mammary tumours also differed little between groups. While the number of preneoplastic mammary hyperplastic alveolar nodules (HAN) was significantly higher in breeders than in virgins, little difference was observed between groups in the end-bud formation and the duct growth of normal glands and the growth of HAN. These results indicate that in SHN mice virgins have similar mammary tumour potential to breeders, which is much advantageous for practical use of this strain as an animal model for human breast and other types of cancers.

Tumor progression and metastasis in murine D2 hyperplastic alveolar nodule mammary tumor cell lines.

We have examined tumor progression and metastatic properties of three clonal murine mammary tumor cell lines of recent origin (D2A1, D2.OR and D2.1). These lines were derived from spontaneous mammary tumors which originated from a D2 hyperplastic alveolar nodule (HAN) line. D2A1 cells were more malignant than D2.OR or D2.1 cells, whether measured by experimental metastasis assays after intravenous injection in nude mice or chick embryos, in vivo growth rate of primary tumors following mammary fat pad injection in nude mice, or spontaneous metastasis assay from primary tumors growing in mammary fat pads. D2A1 cells also were more invasive in vitro in a Matrigel invasion assay than D2.1 cells, while the D2.OR cells were non-invasive in this assay. The increased invasiveness and malignancy of D2A1 cells were associated with increased levels of mRNA for the cysteine proteinase cathepsin L. Levels of osteopontin (OPN), nm23, int-1 and int-2 mRNAs were also examined. Nm23 levels were highest in the most malignant cell line. These cell lines provide a model for studying the tumorigenic and metastatic ability of mammary tumor cells and offer several advantages: they were cloned from mammary tumors that originate from a common source of preneoplastic cells (D2HAN); they are of relatively recent origin; and they have spontaneously arrived at different stages of tumor progression.

Gene expression screening for specific genes associated with mouse mammary tumor development

The preneoplastic hyperplastic alveolar nodule is a frequent and well-characterized precursor to mammary tumors in the mouse. Although the biological characteristics of the preneoplastic state have been understood for many years, the molecular alterations associated with preneoplasia and tumorigenicity are unknown. We applied the technique of differential display of mRNA to two closely matched cell populations of mammary preneoplasias that differed only in their tumorigenic potential. Two mRNAs were isolated that were overexpressed only in tumorigenic preneoplasias and in tumors and not in normal pregnant mammary gland or in nontumorigenic preneoplasias. Partial nucleotide sequencing indicated that one of the mRNAs had not yet been described, whereas the second mRNA was highly homologous to a relatively uncharacteristic gene termed pT-2. These results illustrate the utility of the differential display method for isolating and identifying uniquely expressed genes from tissues maintained in the microenvironment where tumors arise naturally.

The effect of parity, tumor latency and transplantation on the activation of int loci in MMTV-induced, transplanted C3H mammary pre-neoplasias and their tumors.

Mouse mammary tumor virus (MMTV) infection of mammary glands results in proviral insertion into host DNA and activation of cellular genes. Clonal expansion of cells bearing insertional mutations results in hyperplastic alveolar nodules (HAN) and tumors. HAN, transplanted into epithelium-cleared mammary fat pads, form hyperplastic alveolar outgrowths (HOGs). Previous work indicates the commonly MMTV-activated genes wnt-1 and int-2 are rarely affected in HOGs and HOG-derived tumors. To determine the basis of the dichotomy between the frequency of wnt/int gene activation in HOG-derived tumors and tumors from breeders of the identical inbred mouse strain, we compared the activation of wnt-1, int-2 and int-3 in tumors from virgin and breeding C3H mice, in consecutive primary tumors arising in individual C3H breeders and in C3H HOGs at early passages. Activation of wnt-1 or int-2 was rare in HOG-derived tumors (6% and 0%) compared with primary tumors in breeders (52% and 14%). int-3 was never found to be activated. wnt-1 was activated in the same percentage of primary tumors from virgins as from breeders. int-2 was activated only in tumors from breeders. wnt-1 activation also did not correlate with shorter tumor latency in multiple tumors from individual breeders. wnt-1 RNA was not detected in HOGs at early transplant generations, however, low levels of wnt-1 RNA were variably found in the epithelium of virgin mammary glands. We cannot explain why C3H HOGs and their derivative tumors develop without wnt-1 expression when the majority of C3H primary mammary tumors possess an MMTV-activated wnt-1 gene.

Correlation of hyaluronic acid accumulation and the growth of preneoplastic mammary cells in collagen: a longitudinal study.

Hyaluronic acid accumulation is characteristic of mammary tumor cells, and the amount that accumulates seems to correlate with the degree of malignancy of the producing cells. We have tested directly the relationship between hyaluronic acid accumulation and the replication rate of preneoplastic mammary cells in culture. We used nontumorigenic but immortal CL-S1 mouse mammary cells that were derived from a hyperplastic alveolar nodule. Using a collagen gel culture system, we found clear differences in the growth properties of cells before and after Passages 68 to 70. Late passage cells replicated earlier and faster than early passage cells in collagen and on plastic. The rate of cycling resembled that of tumorigenic mouse mammary cells during the first week of culture. Cells seeded at low densities cycled faster than those seeded at high densities during the second week in culture. Exogenous hyaluronic acid, at 10 to 1000 micrograms/ml, neither enhanced nor inhibited CL-S1 cell growth significantly in collagen, regardless of passage. However, by the third day in collagen, late passage cells produced 7 times more total glycosaminoglycans and 12 times more hyaluronic acid per cell than did early passage cells. Late passage cells also deposited 12 times more labeled hyaluronic acid in the matrix than did early passage cells, on a per-cell basis. After a decline in the deposition of hyaluronic acid in the extracellular matrix, growth ceased. The late passage cells did not grow in soft agar, indicating that they had not become neoplastic spontaneously during passage. However, their accelerated growth rate, coupled with the synthesis and secretion of large amounts of hyaluronic acid into the extracellular matrix, may characterize a distinct step in tumor progression in preneoplastic CL-S1 cells.

Analysis of distribution of tumor- and preneoplasia-infiltrating lymphocytes using simultaneous Hoechst 33342 labeling and immunophenotyping.

Hoechst 33342 in vivo staining was combined with immunofluorescent staining of cell surface antigens to quantify the distribution, relative to blood supply, of lymphocytes in a preneoplastic mammary lesion, the murine C4 hyperplastic alveolar nodule (HAN), and the C4 adenocarcinoma which develops from C4 HAN. The vascular supply to lymphocytes expressing Thy 1.2, L3T4, Ly2, and ASGM1 cell surface antigens was evaluated in both tissues. The distribution of ASGM1+ cells, which include natural killer cells, differed between the two tissues, being significantly increased in the 20% brightest Hoechst-stained lymphocyte fraction in HAN but not in C4 tumor. Distribution of T lymphocytes did not differ between the two tissues. The combination of in vivo Hoechst 33342 with in vitro immunofluorescence provides a simple method to evaluate the distribution with regard to blood supply of lymphocyte subsets in solid tumors and preneoplastic lesions.

Estrous cycle dependence of nitrosomethylurea (NMU)-induced preneoplastic lesions in rat mammary gland

Virgin 50-55-day-old rats exhibiting regular estrous cycles were injected i.v. with the direct acting carcinogen 1-nitroso-1-methylurea (NMU) on the morning of proestrus, estrus, or diestrus. Rats were killed at weekly intervals following NMU administration to identify source and number of microscopically identifiable dysplasias. Terminal end bud (TEB) abnormalities appeared within 1 week following NMU administration, with a significantly greater number of abnormal TEBs in mammary glands of rats injected on proestrus (PE) and estrus (E) than on diestrus (DE). Ductal (DH) and ductal alveolar hyperplasias (DAH) and hyperplastic alveolar nodules (HAN) appeared during week 3, with significantly more of each type of lesion appearing by 6 weeks after NMU injection. HAN were most numerous in glands from rats injected on estrus. Adenocarcinomas arose from both the proximal and distal ductal network; at 10 and 12 weeks post NMU, significantly more tumors were found in rats injected on proestrus than diestrus and estrus. These results support the theory that the hormonal environment at the time of NMU administration significantly alters early development of mammary tumors in the rat.

Neoplastic transformation and DNA damage of mouse mammary epithelial cells by N-methyl- N′-nitrosourea in organ culture

An appropriate in vitro system was used to study the effect of a direct-acting carcinogen on the transformation of mammary epithelial cells in the organ culture of the whole mammary gland in vitro. Studies were done to determine the ability of N-methyl-N′-nitrosourea (MNU) to transform the mammary cells in organ culture. Mouse mammary glands were treated with single or multiple doses of MNU during various periods of the culture. To assay for neoplastic transformation potential of MNU on mammary cells, mammary glands were dissociated and the cells were injected into the parenchyma-free inguinal mammary fat pad of syngeneic virgin female host mice. Palpable tumors were observed in injected glands of 23% of the mice after 3–4 months and an additional 31% showed serially transplantable hyperplastic alveolar nodules (HANs). Histopathologic examination of the tissues showed that the tumors were mammary adenocarcinoma. All tumors and hyperplasias were secondarily transplanted into syngeneic animals, resulting in tumors and hyperplasias of similar histopathology. In addition, DNA damage of the epithelial cells in organ culture caused by MNU was also measurable using the new nick translation assay. The most extensive DNA damage occurred when the glands were treated on day 4 and day 5 of the mammogenic culture period. These results demonstrate that the mouse mammary epithelial cells are susceptible to the carcinogenic action of the direct-acting carcinogen MNU and that the whole mammary gland culture system offers an appropriate in vitro model for studying the mechanism of carcinogenesis induced by MNU.

Two-dimensional gel analysis of polypeptides from normal, preneoplastic and neoplastic mouse mammary tissues.

High-resolution two-dimensional polyacrylamide gel electrophoresis (PAGE) was employed to reveal tumor-associated polypeptide changes, using the BALB/c C4 line mouse mammary model system, for which phenotypic and immunogenic alterations accompanying tumor progression are well defined. In the first set of experiments, polypeptide patterns from 20 micrograms whole tissue lysates of normal mammary gland, C4 preneoplastic hyperplastic alveolar nodule outgrowth (HAN) and spontaneous tumor from C4 HAN were compared. In order to normalize for differential cellularity and extracellular protein content in the whole tissues, our analysis included polypeptide patterns from serum, increased concentration of protein from whole normal mammary gland, and primary cultures of epithelial cells from normal gland, HAN and tumor. Using a computer-based image-analysis system, 90 polypeptides were identified in C4 tumor that were absent in C4 HAN, normal mammary gland and serum. None of the 90 polypeptides could be shown to represent a definite qualitative change in the protein composition of tumor epithelium as they were found to be either present in a higher concentration of protein from whole normal gland, or present in the primary epithelial culture from HAN, or absent in the primary epithelial culture from tumor. Conversely in the second set of experiments, when epithelial cultures were used as the starting point for comparisons to locate tumor-associated polypeptides, none of the 15 polypeptides that were present in cultures from three different tumors, and absent in the culture from normal mammary gland was specific to C4 tumor, as they were present in whole tissues of normal gland. Thus our experimental approach detected significant quantitative but no qualitative polypeptide changes in whole tumor tissue, or in tumor-derived epithelial cell cultures. This finding may reflect the limitations of the two-dimensional PAGE method, and warrants caution in the use of such gel analysis alone to identify tumor-associated proteins.

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.

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.

Influence of chemopreventive agents on estradiol metabolism and mammary preneoplasia in the C3H mouse

The C3H strain of mouse has a high incidence of murine mammary tumor virus-induced mammary tumors, and tumorigenesis progresses via the intermediate formation of the preneoplastic, hyperplastic alveolar nodules (HANs). The C3H mouse also exhibits an elevation in 16α-hydroxylation of estradiol which remains unaltered in relation to the age or presence of tumor, but which is detectable well before the emergence of overt mammary cancer. This metabolic pathway leads to the formation of 16α-hydroxyestrone (16α-OHE 1), a putative promoter of mammary cancer. The present study examines the effect of two prototype chemopreventive agents, tamoxifen (TAM) and N-(4-hydroxyphenyl)retinamide (HPR), on 16α-hydroxylation of estradiol and on the growth of HANs. Treatment with TAM, HPR, or a combination of TAM and HPR for 4 weeks in 6- to 8-week-old C3H mice resulted in a consistent elevation in the 16α-hydroxylotion pathway of estradiol metabolism relative to the placebo control group (20.50% ± 2.35%, 21.46% ± 1.49%, 18.00% ± 7.75%, and 12.64% ± 1].45% SD, respectively) and in a significant decrease in the mean frequency of HANs per mammary gland (1.4, 2.1, 0.0, and 5.8, respectively). Mice without any experimental manipulation exhibited an age-dependent progressive increase in HAN frequency from 1.5 per gland at 4 weeks of age to 12.1 per gland at 24 weeks of age. Administration of TAM, HPR, or a combination of TAM and HPR up to 22 weeks of age resulted in a continued suppression of HAN frequency, and the two agents in combination exerted an additive effect on the suppression of HAN development. These results indicate that the promoting effect of 16α-OHE 1 on mammary preneoplasia can be overridden by TAM and HPR, possibly due to the greater affinity of TAM relative to 16α-OHE 1 for the cellular estrogen receptor, or to HPR-induced reduction in 16α-OHE 1 susceptible target tissue (i.e., suppression of HANs).

DDD/1-<I>Mtv-2/Mtv-2</I>コンジェニックマウスとDDD/1マウスの間での乳癌発生に関わる背景因子の比較

To elucidate the possible participation of the Mtv-2 gene in the background factors relevant to mammary tumorigenesis, DDD/1-Miv-2/Mtv-2 (DDD-Mtv-2) congenic and DDD/1 background mice were compared for endocrine and immune organs, mammary gland development, expression of mouse mammary tumor virus (MMTV)-gp 52 antigen, hyperplastic alveolar nodules (HAN) and mammary tumor incidence. The congenic strain had been established by introducing Mtv-2 from GRS/AJms (GR) into DDD/1 mice by repeated 12 backcrosses. Body, thymus, spleen, uterus, ovary, adrenal and pituitary weights, histology of the ovary and mammary gland development showed no differences ascribable to Mtv-2 between both strains of mice at 4,6 and 12 months of age. In contrast, MMTV-gp 52 antigen expression, HAN and mammary tumor occurred in DDD-Mtv-2 but not in DDD/1 mice. These results showed that the Mtv-2 gene stimulates mammary tumorigenesis by constitutional production of endogenous MMTV which transforms mammary epithelial cells but not by influencing the background factors relevant to mammary tumorigenesis. Furthermore, the expression of Mtv-2 appeared to be lower on the DDD/1 than on the GR background.

Spontaneous progression of hyperplastic outgrowths of the D1 lineage to mammary tumors: Expression of mouse mammary tumor virus and cellular proto‐oncogenes

Mammary cancer in mice is characterized by progression through defined stages of preneoplasia, with the most common preneoplastic stage being the hyperplastic alveolar nodule (HAN). We determined the relative levels of RNA expression of various cellular proto-oncogenes and endogenous mouse mammary tumor virus genes in outgrowths and tumors of three sublines of the transplantable D1 HAN preneoplastic outgrowth line. The three sublines differed in relative tumor-producing capabilities. Subline D1B produced a high incidence of tumors with short latency periods, whereas sublines D1C and D1D produced low incidences of tumors with long latency periods. No consistent alteration in proto-oncogene expression correlated with relative tumorigenicity, although tumors frequently contained higher levels of one or more proto-oncogene transcripts as compared with preneoplastic tissue. Slightly elevated (2- to 6-fold) levels of different oncogene transcripts were detected in 13 of 17 tumors as compared with outgrowth tissue, including abl (2 tumors), fps (5 tumors), Ha-ras (6 tumors), and Ki-ras (8 tumors). One tumor contained 45 times more Ki-ras-specific RNA than outgrowth tissue because of a comparable amplification of Ki-ras DNA sequences. Elevated levels of Ha-ras occurred more frequently in tumors of a high-incidence subline than in a less-aggressive subline (5/10 vs 1/7), but this difference was not statistically significant. However, consistent changes in MMTV expression accompanied progression from preneoplastic tissues to mammary tumors. All 17 tumors displayed reduced levels of the MMTV-specific long terminal repeat (LTR) transcript (1.6 kb) as compared with HAN tissue; tumors with moderate levels of LTR transcript expressed the 3.8-kb envelope message as well, one not detected in HANs. Expression of the LTR transcript is apparently influenced by factors in addition to the methylation status of endogenous mouse mammary tumor virus genes, which was similar in outgrowths and tumors. As the survey of representative proto-oncogenes failed to identify a uniform change between HAN and tumors, it is likely that other genes are involved in tumor progression in the mammary gland.