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G Shyamala

Publications and source records attributed to G Shyamala.

At least 19 recordsLinked to original sources
PubMed2005

Proliferation of estrogen receptor-alpha-positive mammary epithelial cells is restrained by transforming growth factor-beta1 in adult mice.

Transforming growth factor (TGF)-beta1 is a potent inhibitor of mammary epithelial proliferation. In human breast, estrogen receptor (ER)-alpha cells rarely co-localize with markers of proliferation, but their increased frequency correlates with breast cancer risk. To determine whether TGF-beta1 is necessary for the quiescence of ER-alpha-positive populations, we examined mouse mammary epithelial glands at estrus. Approximately 35% of epithelial cells showed TGF-beta1 activation, which co-localized with nuclear receptor-phosphorylated Smad 2/3, indicating that TGF-beta signaling is autocrine. Nuclear Smad co-localized with nuclear ER-alpha. To test whether TGF-beta inhibits proliferation, we examined genetically engineered mice with different levels of TGF-beta1. ER-alpha co-localization with markers of proliferation (ie, Ki-67 or bromodeoxyuridine) at estrus was significantly increased in the mammary glands of Tgf beta1 C57/bl/129SV heterozygote mice. This relationship was maintained after pregnancy but was absent at puberty. Conversely, mammary epithelial expression of constitutively active TGF-beta1 via the MMTV promoter suppressed proliferation of ER-alpha-positive cells. Thus, TGF-beta1 activation functionally restrains ER-alpha-positive cells from proliferating in adult mammary gland. Accordingly, we propose that TGF-beta1 dysregulation may promote proliferation of ER-alpha-positive cells associated with breast cancer risk in humans.

Animals↗
PubMed2004

Polymerase chain reaction to detect Chlamydia trachomatis and adenovirus in the nasopharyngeal aspirates from paediatric patients with lower respiratory infections.

Polymerase chain reaction (PCR) was evaluated to detect Adenoviruses and Chlamydia trachomatis on nasopharyngeal aspirates (NPA) obtained 4-5 days after the onset of lower respiratory tract illness in children. Forty-five nasopharyngeal aspirates (NPA) from 45 children with lower respiratory tract infections were processed for the detection of C. trachomatis and Adenovirus by Fluorescent antibody test (FAT), culture and PCR for the cryptic plasmid of C. trachomatis and the gene coding for hexon of Adenoviruses. Seven (13.3%) and 4 (6.6%) of the 45 specimens were positive for C. trachomatis and adenovirus by PCR respectively, which included one specimen each positive for these agents. Cultures were negative for both the organisms. PCRs showed a statistically significant (McNemar test--p= 0.004) higher sensitivity. PCR test is necessary to detect C. trachomatis and adenovirus in nasopharyngeal aspirates obtained 4-5 days after the onset of illness.

Adenovirus Infections, Human↗
PubMed2003

Mammary epithelial cells of PR-A transgenic mice exhibit distinct alterations in gene expression and growth potential associated with transformation.

Expression of the 'A' and 'B' forms of progesterone receptor (PR), in an appropriate ratio is critical for normal mammary development. As such, mammary glands of PR-A transgenic mice, carrying additional 'A' form of PR as transgene, exhibit morphological and histological characteristics associated with transformation. Accordingly, in the present studies, we analyzed these mammary glands for the presence of transformed epithelial cells by examining for alterations in gene expressions and growth potential, known to be associated with different stages of transformation. These studies reveal that, in the aberrant mammary epithelial structures, there is a decrease in p21 expression, an increase in cyclin D1 expression accompanied by an increase in cell proliferation, and a decrease in estrogen receptor alpha (ER alpha). In mammary ducts with normal histology, there is a decrease in p21 expression without an elevation in cyclin D1 expression or cell proliferation or a decrease in ER alpha expression. Treatment of PR-A transgenics with anti-progestin, mifepristone, has no effect on cell proliferation, cyclin D1 or ER alpha expression in the aberrant epithelial structures. In contrast, mifepristone restored the loss of p21 expression in the epithelial cells of both the ducts with normal histology and aberrant structures. Parallel studies reveal no apparent differences between the mammary glands of wild-type and PR-B transgenic mice, which carry additional PR 'B' form. Accordingly, we conclude that (i) mammary glands of PR-A transgenics contain at least two distinct populations of transformed epithelial cells, (ii) the epithelial cell population in the ducts with normal histology contain presumptive immortalized cells, indicative of early stages of transformation, (iii) the aberrant epithelial structures contain later stages of transformation associated with hyperplasias/pre-neoplasias and (iv) the transformation of mammary epithelial cells in PR-A transgenics might be due to a misregulation in progesterone action resulting from overexpression of PR 'A' form.

Animals↗
PubMed2003

Spontaneous pituitary abnormalities and mammary hyperplasia in FVB/NCr mice: implications for mouse modeling.

The FVB/N mouse strain is widely used in the generation of transgenic mouse models. We have observed that mammary glands of wild-type virgin female FVB/NCr mice frequently have the morphologic and histologic appearance of a gland during pregnancy. By 13 months of age, the mammary glands of more than 40% of the mice examined had lobuloalveolar hyperplasia that was characterized by the presence of secretory alveoli and distended ducts apparently containing secretory material. The prevalence of this phenotype further increased with age. The mammary phenotype was highly correlated with the presence of proliferative, prolactin-secreting lesions in the pituitary gland. In mice aged 18 to 23 months, hyperplasia of the pars distalis was seen in 11 of 21 mice (52%), and a further 4 of 21 mice (19%) had pituitary adenomas. Pituitary hyperplasia was already evident in some mice as young as nine months. The pituitary phenotype was also associated with high prevalence (4/6 mice) of spontaneous mammary tumors in aged multiparous, but not virgin FVB/NCr mice. This high prevalence of pituitary abnormalities and their effects on the mammary gland have important consequences for the interpretation of new phenotypes generated in transgenic models using this mouse substrain.

Animals↗
PubMed2003

Persistent mammary hyperplasia in FVB/N mice.

The inbred FVB/N mouse strain is widely used for creating transgenic mice. Over the past decade, persistent mammary hyperplasia has been detected in many multiparous FVB/N female mice sent to the University of California, Davis (UCD) Mutant Mouse Pathology Laboratory (MMPL) by a number of different laboratories. However, the experimental details concerning most specimens were not always available. To confirm these empiric findings, experiments were carried out to evaluate the mammary glands of FVB/N mice under controlled conditions. Persistent mammary hyperplasia that related to parity was found. Weeks after their first to fourth pregnancy, 10 FVB/N female mice from the Lawrence Berkeley National Laboratory (LBNL) colony were studied and the mammary glands were evaluated. The percentage of fat pad filled was estimated, using image analysis. Serum samples and the pituitary gland from other FVB/N mice from the LBNL were assayed for prolactin concentration. Multiparous FVB/N females consistently had persistent mammary hyperplasia. Four of seven females in the LBNL colony had hyperplasia after three pregnancies. A few foci of squamous nodules and sporadic carcinomas also were observed. Thus, some FVB/N females may have persistent mammary hyperplasia after three pregnancies without detectable pituitary abnormalities. Mammary carcinomas also may develop sporadically. These background phenotypes must be considered when interpreting the effect of genetic manipulation in FVB/N mice.

Animals↗
PubMed2002

Pathway pathology: histological differences between ErbB/Ras and Wnt pathway transgenic mammary tumors.

To study phenotype-genotype correlations, ErbB/Ras pathway tumors (transgenic for ErbB2, c-Neu, mutants of c-Neu, polyomavirus middle T antigene (PyV-mT), Ras, and bi-transgenic for ErbB2/Neu with ErbB3 and with progesterone receptor) from four different institutions were histopathologically compared with Wnt pathway tumors [transgenes Wnt1, Wnt10b, dominant-negative glycogen synthase kinase 3-beta, beta-Catenin, and spontaneous mutants of adenomatous polyposis coli gene (Apc)]. ErbB/Ras pathway tumors tend to form solid nodules consisting of poorly differentiated cells with abundant cytoplasm. ErbB/Ras pathway tumors also have scanty stroma and lack myoepithelial or squamous differentiation. In contrast, Wnt pathway tumors exhibit myoepithelial, acinar, or glandular differentiation, and, frequently, combinations of these. Squamous metaplasia is frequent and may include transdifferentiation to epidermal and pilar structures. Most Wnt pathway tumors form caricatures of elongated, branched ductules, and have well-developed stroma, inflammatory infiltrates, and pushing margins. Tumors transgenic for interacting genes such as protein kinase CK2alpha (casein kinase IIalpha), and the fibroblast growth factors (Fgf) Int2/Fgf3 or keratinocyte growth factor (Kgf/Fgf7) also have the Wnt pathway phenotype. Because the tumors from the ErbB/Ras and the Wnt pathway are so distinct and can be readily identified using routine hematoxylin and eosin sections, we suggest that pathway pathology is applicable in both basic and clinical cancer research.

Animals↗
PubMed2002

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.

Aging↗
PubMed2001

PR localization and anterior pituitary cell populations in vitro in ovariectomized wild-type and PR-knockout mice.

The PR is critical for normal female reproductive function in mammals, including primates, and the PR-knockout mouse is an important model for establishing PR targets. For example, LH secretion is significantly altered both in vivo and in vitro in female PR-knockout mice, but to establish specific mechanisms affected by the absence of the PR in the mouse requires characterization of wild-type mouse cell biology. As steps toward this, the aims were to establish whether altered LH secretion in PR-knockout mice reflects altered mouse gonadotrope cell lineage during development secondary to PR deletion and to test the assumption that PR in wild-type mouse pituitaries has the same exclusive gonadotrope localization and E2 and progesterone regulation as in rat and monkey pituitaries. As an in vitro model, dispersed pituitary cells from 2-wk ovariectomized wild-type or PR-knockout mice were cultured +/- E2 for 3 d. These cells were subjected to dual immunofluorescence staining for PR and LH, PRL, or GH. The proportion of LH-gonadotropes (8-9%) and somatotropes (26-29%) was not different for PR-knockout and wild-type cultures with or without E2. Lactotrope composition (41-42%) was the same in wild-type and PR-knockout, and E2 resulted in a similar and significant increase in the proportion (57-59%) for both mouse types. Nuclear PR immunoreactivity was absent in all PR-knockout pituitary cells. For wild-type, all LH gonadotropes showed nuclear PR immunoreactivity that was up-regulated by E2 (>10-fold increase). Progesterone exposure for 10 h but not 3 h led to a 40% decrease in PR immunoreactivity in LH-gonadotropes. Unexpectedly, PR immunoreactivity also localized to all lactotropes and was up-regulated by E2 and down-regulated by progesterone. In summary, the absence of PR has no effect on the proportion of LH gonadotropes, lactotropes, and somatotropes in ovariectomized PR-knockout mouse pituitary cultures. For ovariectomized wild-type mice, gonadotropes in the in vitro model contain PR that is up-regulated by E2, but the downregulation by progesterone is modest, compared with that previously reported for an in vitro rat model. In contrast to rats and monkeys, E2-dependent PR also is present in lactotropes of ovariectomized wild-type mice. These results underscore the risks in assuming identical cell biology between rats and mice.

Animals↗
PubMed2000

Ductal growth is impeded in mammary glands of C-neu transgenic mice.

The steroid hormone, estradiol, is essential for both the growth of normal breast and induction of mammary carcinomas. The growth promoting effects of estrogen are presumed to be mediated by growth factors, in particular, epidermal growth factor, which mediates its effects through erbB receptors, erbB1 and erbB2/C-neu. C-neu is amplified and over-expressed in a large number of human cancers and transgenic mice over-expressing C-neu also develop mammary tumors. However, as yet, the impact of C-neu over-expression on estrogen action during normal mammary development and hence, its precise role in carcinogenesis, remains unclear. In the present studies, we demonstrate that estradiol-dependent mammary ductal growth accompanying puberty is impaired in transgenic mice expressing wild type Cneu, and is intrinsic to the tissue. The impairment is not due to an overall impairment in estrogen action, since progesterone receptor expression is unaffected in C-neu mice. It is also not due to an intrinsic inability of the epithelial cells to proliferate, since impeded ductal growth co-exists with alveolar growth during pregnancy. Therefore, we propose that, depending on the physiological state, C-neu may either promote or inhibit the growth of mammary epithelial cells, and discuss its potential significance to carcinogenesis.

Aging↗
PubMed2000

Impact of progesterone receptor on cell-fate decisions during mammary gland development.

Mammary epithelium contains lineage-limited progenitors that give rise to cells that form distinct morphological structures, ducts vs. lobules, depending on the endocrine status of the female. Progesterone signaling through progesterone receptor (PR) is essential for lobulo-alveolar development that accompanies pregnancy, but not for ductal growth accompanying puberty. PR exists in two molecular forms, A and B, and an imbalance in the native ratio of the two isoforms can lead to alterations in PR signaling. Indeed, as we reported previously, in transgenic mice carrying additional A form of PR, mammary development is abnormal, characterized by excessive lateral ductal branching. This suggests that alterations in PR signaling may have important consequences to mammary development, particularly with regard to ductal vs. alveolar growth. To test this further, we created transgenic mice carrying additional B form of PR and report that mammary development in these mice is also abnormal, characterized by inappropriate alveolar growth. More importantly, these mammary glands, on serial transplantation, undergo a premature arrest in ductal growth without any alteration in the potential for lobulo-alveolar growth. Such an arrest in ductal growth does not occur with transgenics carrying additional A form of PR. These studies, therefore, provide strong evidence to indicate that PR signaling may be of paramount importance for appropriate cell-fate decisions during normal mammary development, and also that this requires a regulated expression of the two isoforms.

Animals↗
PubMed1999

Progesterone signaling and mammary gland morphogenesis.

Progesterone was identified as a mammogenic hormone several years ago but until now its precise role in mammary development has remained obscure. Recently with the generation of several transgenic mouse models and development of reagents for analysis of progesterone receptor expression, the role of progesterone signaling in mammary development is becoming more clear. The most significant observations to emerge from these studies are (1) progesterone receptors (PR) are present in a heterogeneous manner in the epithelial cells and undetectable in the surrounding fat pad; (2) they are essential for lobuloalveolar and not for ductal morphogenesis; (3) progesterone signaling through progesterone receptors, leading to lobuloalveolar development, is initiated in the epithelium and may occur through paracrine mechanisms; and (4) a regulated expression of the two isoforms of progesterone receptor is critical for maintaining appropriate responsiveness to progesterone and hence, epithelial cell replicative homeostasis. These studies also reveal that the consequences of progesterone signaling through progesterone receptor may depend on the cell context, cell-cell and cell-extracellular matrix interactions, the dynamics of PR turnover and the fate of PR positive cells.

Animals↗
PubMed1999

Steroid regulation of progesterone receptor expression in cultured rat gonadotropes.

During the preovulatory period, the pituitary action of progesterone is biphasic, moving from a severalfold augmentation of the gonadotropin release action of GnRH to a suppression of GnRH efficacy, which occurs in rats over a period of about 12 h, but the extent to which these biphasic effects are dependent on alterations in progesterone receptor (PR) expression is not known. To address this, as well as the localization of PR in cultured rat pituitary cells, we used cells from ovariectomized rats cultured +/- 0.2 nM E2 with acute progesterone treatment on day 3. Northern blot of poly(A+) RNA extracts showed multiple PR messenger RNA (mRNA) transcripts between 4.8-10.2 kb; E2 treatment led to a 5- to 6-fold increase in the predominant PR mRNA transcripts (5.1 and 10.1 kb). In the presence of E2, 200 nM progesterone resulted in a decrease in steady-state PR mRNA levels by 3 h of exposure, with the greatest decrease around 6 h (50% of E2 control) and recovery by 12 h. Similarly treated pituitary cultures were subjected to dual immunofluorescence staining for LH and PR. In the absence of E2, PR was undetectable. In the presence of E2, essentially all LH-positive cells were positive for PR and only 1-2% of PR-immunopositive cells were negative for LH, possibly reflecting FSH-exclusive gonadotropes. PR staining was predominantly nuclear, but 20 nM progesterone led to a gradual increase in cytoplasmic staining, with the nuclear-to-cytoplasmic ratio decreasing to near unity by 9-12 h of exposure. In summary, we show for the first time, that PR colocalizes with LH in cultured female rat pituitary cells and that E2 induces expression of PR mRNA, as well as PR protein, in rat gonadotropes. In the presence of E2, progesterone causes a rapid but transient down-regulation of PR message; recovery of PR mRNA is accompanied by an increase in cytoplasmic PR, suggestive of an increase in synthesis. These dynamic changes implicate the gonadotrope PR as having a significant role within the temporal context of the rat preovulatory period.

Animals↗
PubMed1998

Transgenic mice carrying an imbalance in the native ratio of A to B forms of progesterone receptor exhibit developmental abnormalities in mammary glands.

In this report we document the creation of transgenic mice in which the native ratio of A and B forms of progesterone receptor (PR) has been altered by the introduction of additional A form as transgene. We also show that in these mice there is an aberration in mammary development. In ovariectomized prepubertal PR-A transgenic mice, end buds with unusual morphology persist after ovariectomy, and in young adult nonovariectomized mice, mammary glands have extensive lateral branching. The glands of adult mice also exhibit ductal hyperplasia with a disorganized basement membrane and decreased cell-cell adhesion, features commonly associated with neoplasia. Because progesterone is a mitogenic hormone in mammary glands and PR is required for mammary development, these data provide direct evidence that in vivo a regulated expression of the two isoforms of PR is critical for appropriate cellular response to progesterone and that for mammary glands this may have major implications to carcinogenesis.

Animals↗
PubMed1997

Murine 86-kDa heat shock protein gene and promoter.

The class of 90 kDa heat shock proteins (Hsp90) is among the most abundant heat shock proteins (Hsps) in eukaryotic cells. In vertebrates, Hsp90 is encoded by two distinct gene families giving rise to products of 84 and 86 kDa. In mice the expression of these two genes, hsp84 and hsp86, vary with respect to each other in responses to stress, and also in response to signals for growth and development. Therefore, as a step towards understanding the molecular basis for the differential regulation of these two genes, we have isolated and characterized genomic clones of the murine hsp86 gene and its 5' flanking region. The gene is composed of eleven exons interrupted by 10 introns. The 5' region contains consensus TATA, several stimulatory protein-1 binding site (SP1) elements as well as six consensus heat shock elements (HSE) 5' of the transcription start site. An 806 bp fragment of the 5' promoter region conferred constitutive expression upon a reporter gene and this expression was increased upon heat shock.

Animals↗
PubMed1996

Cloning and characterization of the promoter for murine 84-kDa heat-shock protein.

The 90-kDa heat-shock (HS) proteins (HSP90) are members of the HSP family. Their synthesis is inducible by HS and a variety of stress signals. HSP90 is also abundant under normal physiological conditions and its synthesis can be regulated during growth and differentiation. Therefore, HSP90 is speculated to have important biological functions, in addition to its role in mediating stress responses. However, the mechanism(s) regulating hsp90 gene expression in nonstressed cells is poorly understood. As a prerequisite towards understanding the basis for hsp90 regulation, we have cloned and characterized the 5' flanking region of murine hsp84, one of two genes which code for HSP90 proteins. Full basal promoter activity of hsp84 was found to be associated with a 627-bp region immediately upstream from the transcription start point (tsp). Sequence analysis revealed several putative regulatory elements, including a HS element (HSE), an AP1-binding site (AP1), a cyclic AMP response element (CRE), and four stimulatory protein-1-binding sites (SP1). HS inducibility required the HSE which was bound by HS transcription factor-1(HSF-1) present in extracts prepared from cells exposed to HS. The HSE was not required for basal (non-HS) expression, but, interestingly, two protein-HSE complexes, devoid of HSF-1 and HSF-2, were formed under these conditions. The potential significance of these findings to the expression of hsp84 under normal physiological conditions is discussed.

3T3 Cells↗
PubMed1996

Progesterone receptors in the mouse mammary duct: distribution and developmental regulation.

During pregnancy, in response to rising levels of estrogen and progesterone, mammary epithelial cells proliferate and differentiate, giving rise to lobuloalveolar structures from which milk is secreted during lactation. Progesterone, in conjunction with estrogen, also promotes mammary epithelial growth during puberty, but unlike development during pregnancy, this results in ductal rather than secretory structures. Recent studies using mice lacking progesterone receptors indicate that these receptors are essential for secretory development. This suggests that during different phases of normal mammary growth and differentiation, progesterone receptors may mediate their effects through different mechanisms, depending on the physiological state of the animal. Therefore, as a prerequisite for understanding the role of progesterone in normal mammary development, we have investigated the spatial and temporal distribution of progesterone receptor-containing cells. In the mammary epithelium of prepubertal mice, high levels of progesterone receptor mRNA and protein are present in the actively growing end buds and ductal branches as well as in the mature duct. Receptor protein was found in some but not in all epithelial cells, and these cells were characterized by large, round, or oval nuclei containing diffuse chromatin. Cytoplasmic and nuclear immunostaining were observed; interestingly, this staining occurred in separate cells that were in close proximity to each other. The relative proportion of cytoplasmic versus nuclear staining cells changed during pregnancy such that in this developmental state, the nuclear staining cells predominated. The potential significance of these observations as they pertain to normal mammary epithelial growth and differentiation are discussed.

Animals↗