Induction of squamous metaplasia: requirement for cell multiplication, and competition with lobuloalveolar development in cultured mammary glands

Mouse mammary glands were previously shown to undergo either of two courses of development and differentiation in whole organ culture. The combination of insulin, prolactin, aldosterone, and hydrocortisone induces a structural development of lobuloalveoli, followed by casein production. In the second course, the mixture of dibutyryl cyclic AMP, prostaglandins E1, E2 and B1, and papaverine brings about an extensive squamous metaplasia and excessive keratinization. In the present study, the foci of the metaplastic squamous cells appeared to originate from single or very few cells. A preferential stimulation of squamous cell multiplication was involved in the induction process. Twice the relative number of nuclei incorporated 3H-thymidine in the squamous metaplastic cells than in the surrounding cuboidal epithelium, according to autoradiography. The necessity for cell multiplication was indicated by the reversible and complete inhibitions of both the metaplastic squamous development and 3H-thymidine incorporation by 1 mM hydroxyurea in the culture medium. Simultaneous inductions of both courses of development and differentiation revealed a competitive and reciprocal relationship between the two pathways. The concurrent expressions of both courses were considerably less than those achieved when either pathway was induced alone. Only the combination of the three types of inducers of squamous metaplasia was able to compete effectively with the hormonal induction of lobuloalveolar development and differentiation. The findings suggest that individual metaplastic squamous foci may originate as clones of cells by processes that require cell multiplication, rather than through a direct non- replicative conversion of pre-existent cells of the cuboidal epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cortactin-binding protein 90 (CBP90) expression in the mouse mammary glands during prolactin-induced lobuloalveolar development

We have previously performed suppression subtractive hybridization to identify genes that were induced during prolactin (PRL)-driven lobuloalveolar development of the mammary gland. This suggested that cortactin-binding protein 90 (CBP90), which is known to be a brain-specific protein that binds to cortactin, was expressed under the regulation of PRL in the mammary glands (preliminary observation). In this study, the expression of CBP90 was examined in the mammary glands of mice under manipulated hormonal circumstances. PRL treatment by 9 days of pituitary grafting induced CBP90 expression in the normal mammary glands but not in the cleared fat pads, while cortactin was expressed constitutively in both the normal mammary glands and the cleared fat pads. Unlike milk proteins, longer treatment with PRL (36 days of pituitary grafting) did not increase the expression level of CBP90 mRNA, while it slightly increased the cortactin mRNA level. Mammary CBP90 mRNA expression was induced by pituitary grafting but not by progesterone treatment in PRL-deficient mice, while pituitary grafting induced mammary CBP90 expression in ovariectomized PRL-deficient mice only when estrogen and progesterone were appropriately supplemented to permit the formation of alveolar buds. The CBP90 protein was detected by immunohistochemistry in the luminal epithelium of the alveolar buds and more faintly in the ductal epithelium. Thus, from the unique expression pattern, CBP90 may be useful as a molecular marker for the hormone-stimulated development of mammary alveolar buds.     

Generation of lobuloalveolar development from isolated rat mammary ducts and end buds

Implantation of excised bud-free ductal fragments (DUCTS), terminal end buds (TEBs), or alveolar buds (ABs) from virgin mammary glands of Wistar-Furth rats into interscapular fat pads of syngeneic female rats produces, after 16 weeks, complete ductal outgrowths including TEBs and ABs. Treatment of the recipient rats with perphenazine for 1 day or mating them after 12 weeks and then isolating the resultant outgrowths after 16 weeks produces significantly larger outgrowths than those from untreated hosts. The outgrowths consist of distended ducts and lobules or distended ducts and alveoli, respectively. Histochemical and immunocytochemical staining of the outgrowths with reagents that depict epithelial, myoepithelial, and lactating alveolar cells (peanut lectin alone, monoclonal and polyclonal antibodies to rat caseins) indicate similar cell compositions and arrangements for all outgrowths irrespective of their source; these are also similar to the mammary glands of the perphenazine-stimulated or lactating hosts. There is one major difference: the degree of staining of peanut lectin alone and the anti-caseins is greater for outgrowths produced by the ABs and TEBs than for those produced by the DUCTs. DUCT implants left for 1 year after cessation of lactation of the hosts are still stained appreciably by peanut lectin alone and by the anti-caseins, particularly the luminal secretions. Therefore, the complete morphogenetic and cell differentiating ability for generating mammary glands is present in bud-free ducts, but this ability can be enhanced in TEBs/ABs or abnormally expressed at ectopic sites.     

Constant light exposure induces damage and squamous metaplasia in Harderian glands of albino mice

Harderian glands from control albino mice kept in a cyclical light/dark environment had tubulo-alveoli comprised of lipid-filled glandular epithelial cells. The porphyrin content of the gland measured 122 μg/100 mg gland. Constant light exposure for 24 hr caused exopthalmos grossly. Histologically most of the secretory cells were swollen and the lumens of many tubulo-alveoli were obliterated; a few areas of the gland showed damage. The porphyrin content had decreased to 116 μg/100 mg gland. After 3 days of constant light exposure the tubulo-alveoli were markedly altered. Lipid and cellular debris filled the lumens, and lining cells were highly irregular, ranging in shape from columnar to squamous. The porphyrin content had decreased to 72 μg/100 mg gland and leukocytes and macrophages were abundant. Despite this extensive damage a number of tubulo-alveolar epithelial cells were observed undergoing mitosis. After 7 days of constant exposure to light, some tubulo-alveolar epithelial cells had undergone squamous metaplasia, and the porphyrin content had dropped markedly to 50 μg/100 mg gland. These pronounced cellular changes are believed to result from a direct effect of light on the gland.     

Local effects of EGF, alpha-TGF, and EGF-like growth factors on lobuloalveolar development of the mouse mammary gland in vivo

Five-week-old female mice supplemented with estradiol and progesterone are able to respond to epidermal growth factor (EGF) and EGF-like growth factors (alpha-transforming growth factor [alpha-TGF] and crude mammary-derived growth factor) with local lobuloalveolar development when these growth factors are directly introduced into the mammary glands via slow-release cholesterol-based pellets. Contralateral glands receiving pellets containing only cholesterol showed no growth response. The local growth effect is maximal at 4-5 days of exposure to hormones and growth factors. The glands appear to be more sensitive to alpha-TGF than EGF, since local development is seen with one-fifth the level of the former vs. the latter growth factor and can be seen even in the absence of the systemic estrogen/progesterone supplement.     

Squamous cell carcinoma and mammary abscess formation through squamous metaplasia in Smad4/Dpc4 conditional knockout mice

Smad4 is a central mediator for TGFbeta signals, which play important functions in many biological processes. To study the role of Smad4 in mammary gland development and neoplasia, we disrupted this gene in mammary epithelium using a Cre-loxP approach. Smad4 is expressed in the mammary gland throughout development; however, its inactivation did not cause abnormal development of the gland during the first three pregnancies. Instead, lack of Smad4 gradually induced cell proliferation, alveolar hyperplasia and transdifferentiation of mammary epithelial cells into squamous epithelial cells. Consequently, all mutant mice developed squamous cell carcinoma and/or mammary abscesses between 5 and 16 months of age. We demonstrated that absence of Smad4 resulted in beta-catenin accumulation at onset and throughout the process of transdifferentiation, implicating beta-catenin, a key component of the Wnt signaling pathway, in the development of squamous metaplasia in Smad4-null mammary glands. We further demonstrated that TGFbeta1 treatment degraded beta-catenin and induced epithelial-mesenchymal transformation in cultured mammary epithelial cells. However, such actions were blocked in the absence of Smad4. These findings indicate that TGFbeta/Smad4 signals play a role in cell fate maintenance during mammary gland development and neoplasia.     

The epithelial glucocorticoid receptor is required for the normal timing of cell proliferation during mammary lobuloalveolar development but is dispensable for milk production

Glucocorticoids have been shown to influence mammary gland function in vivo and to stimulate milk protein gene expression in vitro. Here, we describe the generation and analysis of a mouse model to study glucocorticoid receptor (GR, NR3C1) function in mammary epithelial cells. Using the Cre-loxP system, mutant mice were obtained in which the GR gene is specifically deleted in epithelial cells during lobuloalveolar development, leading to a complete loss of epithelial GR at the onset of lactation. Mice harboring the mammary-epithelial-specific GR mutation are able to nurse their litters until weaning. During pregnancy, however, GR deficiency delays lobuloalveolar development, leading to an incomplete epithelial penetration of the mammary fat pad that persists throughout lactation. We identified a reduced cell proliferation during lobuloalveolar development as reason for this delay. This reduction is compensated for by increased epithelial proliferation after parturition in the mutant glands. During lactation, GR-deficient mammary epithelium is capable of milk production and secretion. The expression of two milk proteins, namely whey acidic protein and beta-casein, during lactation was not critically affected in the absence of GR. We conclude that GR function is not essential for alveolar differentiation and milk production, but influences cell proliferation during lobuloalveolar development.     

Induction of beta-casein synthesis in the mammary glands of rats treated with plant extracts

Water extracts from various african plants reputed to be lactogenic have been administered orally to mature virgin rats. After 4 days of treatment, the presence of beta-casein in mammary gland has been evaluated using a radioimmunoassay. Four out of the 7 plants examined proved to have an authentic capacity to stimulate milk synthesis.     

ErbB4 signaling in the mammary gland is required for lobuloalveolar development and Stat5 activation during lactation.

Signaling by members of the epidermal growth factor receptor family plays an important role in breast development and breast cancer. Earlier work suggested that one of these receptors, ErbB4, is coupled to unique responses in this tissue. To determine the function of ErbB4 signaling in the normal mouse mammary gland, we inactivated ErbB4 signaling by expressing a COOH terminally deleted dominant-negative allele of ErbB4 (ErbB4DeltaIC) as a transgene in the mammary gland. Despite the expression of ErbB4DeltaIC from puberty through later stages of mammary development, an ErbB4DeltaIC-specific phenotype was not observed until mid-lactation. At 12-d postpartum, lobuloalveoli expressing ErbB4DeltaIC protein were condensed and lacked normal lumenal lactation products. In these lobuloalveoli, beta-casein mRNA, detected by in situ hybridization, was normal. However, whey acidic protein mRNA was reduced, and alpha-lactalbumin mRNA was undetectable. Stat5 expression was detected by immunohistochemistry in ErbB4DeltaIC-expressing tissue. However, Stat5 was not phosphorylated at Y694 and was, therefore, probably inactive. When expressed transiently in 293T cells, ErbB4 induced phosphorylation of Stat5. This phosphorylation required an intact Stat5 SH2 domain. In summary, our results demonstrate that ErbB4 signaling is necessary for mammary terminal differentiation and Stat5 activation at mid-lactation.     

Enhancement of squamous cell development in cultured skin by cyclic adenine nucleotide and prostaglandins

The present study tests the hypothesis that agents known to elevate the level of intracellular cyclic adenine nucleotide may direct different epithelial cells onto a pathway of epidermoid (squamous) development and differentiation. We report here that the mixture of dibutyryl cyclic AMP (dbcAMP), prostaglandins E1, E2 and B1 (PG E1, E2, B1), and papaverine (pap) enhances the rate of normal squamous cell development in organ-cultured skin of chick embryos. The three components may act synergistically to elevate the level of intracellular cyclic adenine nucleotide. We recently reported that the same group of agents induces abnormal development (squamous metaplasia) and aberrant differentiation (keratin production) in the normally cuboidal epithelium of cultured whole mammary glands of mice [1]. Thus, dbcAMP, PG E1, E2, B1, and pap are effective in enhancing normal squamous cell development and also in inducing squamous metaplasia de novo in the epithelial components of two different organs of embryonic and adult animals of two classes of vertebrates. The combined findings are suggestive that cyclic adenine nucleotide together with the prostaglandins may act generally on diverse types of epithelia to bring about squamous cell development and a differentiation marked by keratin production.     

The major DNA polymerase in cultured plant cells: Partial purification and correlation with cell multiplication

A DNA polymerase activity was isolated from cells of Oryza sativa L. grown in suspension culture. Molecular mass ( 180,000), optimal requirements for pH (neutral), Mg²⁺ (5–10 mM), Mn²⁺ (1 mM), template preference (activated DNA), lack of activity with native or denatured DNA, and sensitivity to N-ethylmaleimide and ionic strength are similar to those of the vertebrate -polymerase. Like DNA polymerase , the DNA polymerase described in this work is the most abundant in proliferating cells of Oryza sativa L., Parthenocissus tricuspidata (Siebold et Zucc.) Planchon, Acer pseudoplatanus L., and Medicago sativa L. and responds to changes in the rate of cell multiplication. We therefore postulate that this -like DNA polymerase is the replicating enzyme of plant cells.Abbreviations BSA bovine serum albumin - EDTA ethylendiamino-tetracetic acid - DTT dithiothreitol - PTSF p-toluenesulfonyl fluoride     

Cellular basis of urothelial squamous metaplasia: roles of lineage heterogeneity and cell replacement

Although the epithelial lining of much of the mammalian urinary tract is known simply as the urothelium, this epithelium can be divided into at least three lineages of renal pelvis/ureter, bladder/trigone, and proximal urethra based on their embryonic origin, uroplakin content, keratin expression pattern, in vitro growth potential, and propensity to keratinize during vitamin A deficiency. Moreover, these cells remain phenotypically distinct even after they have been serially passaged under identical culture conditions, thus ruling out local mesenchymal influence as the sole cause of their in vivo differences. During vitamin A deficiency, mouse urothelium form multiple keratinized foci in proximal urethra probably originating from scattered K14-positive basal cells, and the keratinized epithelium expands horizontally to replace the surrounding normal urothelium. These data suggest that the urothelium consists of multiple cell lineages, that trigone urothelium is closely related to the urothelium covering the rest of the bladder, and that lineage heterogeneity coupled with cell migration/replacement form the cellular basis for urothelial squamous metaplasia.     

Cellular expression of estrogen and progesterone receptors in mammary glands: regulation by hormones, development and aging

At present, there is an extensive body of literature documenting the participation of estrogen receptors (ER) and progesterone receptors (PR) in mammary gene expression. Yet, the precise roles of these receptors in regulating mammary development, carcinogenesis and the growth of a subset of tumors still remain unclear. Mammary glands are composed of various cell types with different developmental potentials. Further, ultimately, that it is their mutual interactions which dictate the behavior of mammary epithelial cells. Therefore, to resolve the roles of ER and PR in normal mammary growth, differentiation and carcinogenesis, analyses for the expression of these receptors at the level of individual cell types is of paramount importance. Accordingly, in the present studies using immunolocalization techniques, we document the ontogeny and cellular distribution of ER and PR during mammary development and in response to ovarian hormones and aging. In addition, we discuss the potential biological significances of the expression patterns of ER and PR during various physiological states. We believe that the observations reported here should provide a conceptual framework(s) for elucidating the roles of ER and PR in normal and neoplastic mammary tissues.     

Expression of a putative stem cell marker,Musashi 1, in mammary glands of ewes

Several recent studies demonstrated that development, function and remodelling of mammary glands involved multipotent cells, but no specific molecular markers for mammary epithelial stem cells were revealed. These studies principally concerned human and mouse mammary tissue, but mammary stem cells could be a valuable tool in agricultural production and bioengineering in farm animals. The Musashi-1 (Msi 1) gene encodes an RNA binding protein, which is likely to be associated with self-renewal of neural, intestinal and mammary progenitor cells and is believed to influence the Notch signalling pathway. In this study Musashi-1 expression was detected using immunohistochemistry and in situ hybridisation analysis on mammary glands of ewes at different developmental stages. The protein expression was observed in the epithelial cells at all stages examined. In situ hybridization analysis showed that Msi 1 mRNA has an expression pattern similar to the encoded protein, with positive staining in both nuclei and cytoplasm of ductal, secretory and stromal cells. Ultrastructural in situ analysis confirmed the nuclear and cytoplasmatic expression of Msi. Quantitative analysis of Msi 1 gene expression showed a strong correlation with that of Ki-67, that is a marker of cell proliferation. This is the first report outlining expression of Msi 1 in ovine mammary glands during a complete cycle of lactation.     

Growth and development of the mammary glands of livestock: a veritable barnyard of opportunities

The mammary glands of all mammals are rich and diverse in their histomorphogenesis, developmental biology, genomics and metabolism. Domesticated livestock comprise a unique population for the analysis of mammary gland and lactation biology, where much of what has been learned about these topics originates from studies of these species. However, with the strong trend toward using rodents as flexible and attractive models for normal mammary biology and cancer, there is a growing void of new information related to biology of the mammary glands in these relevant and informative domestic livestock. In turn, this trend threatens to reduce opportunities to either capitalize on an abundance of pre-existing data or to apply this information to studies of lactation and cancer. Herein we review the unique and discerning features of mammary gland development in several domestic livestock species including cows, sheep and pigs and provide an overview of the factors regulating it. At the same time we discuss some of the key considerations for studying these species, their limitations, and the associated opportunities. From such an analysis it quickly becomes clear that much remains to be learned about the mammary glands of domestic livestock, particularly given their many similarities to the human breast, the unique biological mechanisms they employ, and the phenotypic variation they afford.     

Progesterone stimulates DNA synthesis and lobulo-alveolar development in mammary glands in ovariectomized mice

The objective of this study was to determine whether sustained progesterone (P) use in the absence of estrogen could influence mammary development in mice. Three-week-old intact or ovariectomized mice were primed with subcutaneous (s.c.) cholesterol (C), estrogen (E), P, or estrogen and progesterone (E/P) together. Nine days after priming, mammary glands were removed and incubated as a whole organ in media supplemented with various combinations of lactogenic hormones. After 5 days in whole organ culture, glands were removed and end buds, alveolar buds and lobulo-alveoli were quantified. Glands from mice primed with C or E developed significantly less lobulo-alveoli than glands from mice primed with P or E/P. While the development was greater in animals treated with E/P compared to those treated with P, it was clear that P in the absence of E could still induce lobulo-alveolar development. We have shown in this paper that P, in the absence of E, can stimulate cell proliferation during priming. Subsequently, the P primed glands can differentiate in response to lactogenic hormones. J. Cell. Physiol. 180:298–304, 1999. © 1999 Wiley-Liss, Inc.     

Utility of HPV DNA detection in thin-layer,liquid-based tests with atypical squamous metaplasia

OBJECTIVE: To determine if testing for HPV is useful in the management of patients with atypical squamous metaplasia (ASM) and to evaluate a small group of patients with atypical squamous cells in the setting of an atrophic cellular profile (estrogen test [ESTT]). STUDY DESIGN: Presence of HPV DNA was determined on 104 ASM and ESTT cases on residual ThinPrep specimens using Hybrid Capture II. Results of the HPV DNA test were correlated with subsequent biopsy or repeat Pap test results. RESULTS: Of 63 patients with ASM on ThinPrep Pap tests, 52% were associated with histologically proven SIL when HPV DNA was detected by Hybrid Capture II. None of the 17 patients who were negative for HPV DNA had subsequent evidence of HPV infection by biopsy or repeat Pap test. Among 14 patients with ESTT, none of the 12 who were negative for HPV DNA had subsequent evidence of HPV infection. CONCLUSION: ASM and ESTT with a negative HPV DNA test can be followed routinely, and a colposcopic examination is not warranted.