RESUMO
We have examined the developmental expression of the murine breast and ovarian cancer susceptibility gene, Brca1, to investigate its role in the control of cell growth and differentiation. Specifically, we have analysed Brca1 expression during embryonic development, in adult tissues, and during postnatal mammary gland development, particularly in response to ovarian hormones. Our results suggest that Brca1 is expressed in rapidly proliferating cell types undergoing differentiation. In the mammary gland, Brca1 expression is induced during puberty, pregnancy, and following treatment of ovariectomized animals with 17 beta-estradiol and progesterone. These observations imply that Brca1 is involved in the processes of proliferation and differentiation in multiple tissues, notably in the mammary gland in response to ovarian hormones.
Assuntos
Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Glândulas Mamárias Animais/metabolismo , Proteínas de Neoplasias/biossíntese , Fatores de Transcrição/biossíntese , Animais , Proteína BRCA1 , Sequência de Bases , Divisão Celular/genética , Células Cultivadas , Desenvolvimento Embrionário e Fetal , Epitélio/metabolismo , Estradiol/farmacologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hibridização In Situ , Lactação , Masculino , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/embriologia , Glândulas Mamárias Animais/crescimento & desenvolvimento , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Oligonucleotídeos Antissenso , Especificidade de Órgãos , Ovariectomia , Paridade , Gravidez , Progesterona/farmacologia , Maturidade Sexual , Fatores de Transcrição/genéticaRESUMO
The observation that normal pathways of differentiation and development are invariably altered during the process of carcinogenesis implies an intrinsic relationship between these processes. This relationship is particularly evident in the breast, as exemplified by the existence of endocrine risk factors for breast cancer that are related to the timing of normal developmental events. Understanding the mechanisms by which normal developmental events alter breast cancer risk is a central focus of our laboratory. Herein, we describe three approaches being taken in our laboratory toward defining the molecular basis of this relationship. These include: determining the roles played by the tumor suppressor genes, BRCA1 and BRCA2, in the normal differentiation and development of the breast; studying the function of three novel protein kinases identified in our laboratory in mammary epithelial development; and defining the molecular and cellular changes that occur in the breast as a result of reproductive events known to influence breast cancer risk.
Assuntos
Neoplasias da Mama/fisiopatologia , Mama/crescimento & desenvolvimento , Reprodução , Animais , Proteína BRCA2 , Neoplasias da Mama/etiologia , Diferenciação Celular/fisiologia , Feminino , Genes BRCA1 , Genes Supressores de Tumor , Humanos , Proteínas de Neoplasias/genética , Fatores de Risco , Fatores de Transcrição/genéticaRESUMO
We have identified a novel protein, BAP1, which binds to the RING finger domain of the Breast/Ovarian Cancer Susceptibility Gene product, BRCA1. BAP1 is a nuclear-localized, ubiquitin carboxy-terminal hydrolase, suggesting that deubiquitinating enzymes may play a role in BRCA1 function. BAP1 binds to the wild-type BRCA1-RING finger, but not to germline mutants of the BRCA1-RING finger found in breast cancer kindreds. BAP1 and BRCA1 are temporally and spatially co-expressed during murine breast development and remodeling, and show overlapping patterns of subnuclear distribution. BAP1 resides on human chromosome 3p21.3; intragenic homozygous rearrangements and deletions of BAP1 have been found in lung carcinoma cell lines. BAP1 enhances BRCA1-mediated inhibition of breast cancer cell growth and is the first nuclear-localized ubiquitin carboxy-terminal hydrolase to be identified. BAP1 may be a new tumor suppressor gene which functions in the BRCA1 growth control pathway.
Assuntos
Proteína BRCA1/metabolismo , Neoplasias da Mama/patologia , Proteínas de Transporte/metabolismo , Tioléster Hidrolases/genética , Tioléster Hidrolases/metabolismo , Proteínas Supressoras de Tumor , Ubiquitina Tiolesterase , Ubiquitina-Proteína Ligases , Sequência de Aminoácidos , Animais , Proteína BRCA1/química , Sequência de Bases , Sítios de Ligação , Neoplasias da Mama/genética , Proteínas de Transporte/biossíntese , Proteínas de Transporte/química , Divisão Celular , Mapeamento Cromossômico , Cromossomos Humanos Par 3 , Feminino , Rearranjo Gênico , Homozigoto , Humanos , Cariotipagem , Neoplasias Pulmonares/genética , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Tioléster Hidrolases/química , Transfecção , Dedos de ZincoRESUMO
Germline mutations in the putative tumor suppressor gene, BRCA1, predispose women to dramatically elevated risks of breast cancer, while germline mutations in the structurally unrelated gene, BRCA2, predispose both men and women to breast cancer. Recent studies have suggested an important developmental role for the murine homologue of BRCA1 in the regulation of proliferation and differentiation. At the present time, however, little is known about the developmental role of BRCA2 or the regulation of its expression in vivo. We have determined the spatial and temporal pattern of expression of the murine homologue of BRCA2 during fetal development, in adult tissues, and in the mammary gland during postnatal development. Our results indicate that Brca2 mRNA expression is highest in proliferating cellular compartments, particularly those undergoing differentiation. In the breast, Brca2 expression is developmentally regulated and is induced during puberty and pregnancy and as a result of parity. Surprisingly, in multiple fetal and adult tissues the spatial and temporal pattern of Brca2 mRNA expression is virtually indistinguishable from that of Brca1, despite the fact that these genes display no homology. These observations suggest that Brca2 is involved in the processes of proliferation and differentiation in the mammary gland and other tissues, and that Brca1 and Brca2 mRNA expression may be regulated by similar pathways and stimuli in multiple cell types. Interestingly, however, our analysis reveals that Brca1 and Brca2 expression are differentially regulated during the development of specific endocrine target tissues, such as the testis during spermatogenesis and the breast during pregnancy. In addition, the ratio of mRNA expression in the mammary glands of adult females relative to adult males is significantly greater for Brca1 than for Brca2. These observations imply that Brca1 and Brca2 mRNA expression are differentially regulated by sex hormones. In order to test this hypothesis, we have analyzed the expression of these two breast cancer susceptibility genes in ovariectomized mice treated with 17beta-estradiol and progesterone. Our results demonstrate that the up-regulation of mRNA expression in the breast by ovarian hormones is significantly greater for Brca1 than for Brca2. These observations suggest that the gender-specific differences in phenotype associated with germline mutations in BRCA2 versus BRCA1 may be related to the differential regulation of these genes by sex hormones.
Assuntos
Diferenciação Celular , Divisão Celular , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genes BRCA1 , Proteínas de Neoplasias/genética , Fatores de Transcrição/genética , Animais , Proteína BRCA2 , Sondas de DNA , Estradiol/farmacologia , Feminino , Hibridização In Situ , Masculino , Glândulas Mamárias Animais/metabolismo , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Proteínas de Neoplasias/biossíntese , Ovariectomia , Progesterona/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/biossínteseRESUMO
We have analyzed the expression of the breast cancer susceptibility gene, Brca2, in mammary epithelial cells as a function of proliferation and differentiation. Our results demonstrate that Brca2 mRNA expression is tightly regulated during mammary epithelial proliferation and differentiation, and that this regulation occurs coordinately with Brca1. Specifically, Brca2 mRNA expression is up-regulated in rapidly proliferating cells; is down-regulated in response to serum deprivation; is expressed in a cell cycle-dependent manner, peaking at the G1/S boundary; and is up-regulated in differentiating mammary epithelial cells in response to glucocorticoids. In each case, an identical pattern of expression was observed for Brca1. These results indicate that proliferative stimuli modulate the mRNA expression of these two breast cancer susceptibility genes. In addition, the coordinate regulation of Brca1 and Brca2 revealed by these experiments suggests that these genes are induced by, and may function in, overlapping regulatory pathways involved in the control of cell proliferation and differentiation.
Assuntos
Proteína BRCA1/biossíntese , Neoplasias da Mama/genética , Mama/citologia , Mama/metabolismo , Regulação da Expressão Gênica , Proteínas de Neoplasias/biossíntese , Fatores de Transcrição/biossíntese , Transcrição Gênica , Animais , Proteína BRCA2 , Neoplasias da Mama/patologia , Diferenciação Celular , Divisão Celular , Linhagem Celular , Células Epiteliais , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Marcadores Genéticos , Humanos , Hidrocortisona/farmacologia , Insulina/farmacologia , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Prolactina/biossíntese , RNA Mensageiro/biossíntese , Células Tumorais CultivadasRESUMO
The susceptibility of the mammary gland to carcinogenesis is influenced by its normal development, particularly during developmental stages such as puberty and pregnancy that are characterized by marked changes in proliferation and differentiation. Protein kinases are important regulators of proliferation and differentiation, as well as of neoplastic transformation, in a wide array of tissues, including the breast. Using a RT-PCR-based cloning strategy, we have identified 41 protein kinases that are expressed in breast cancer cell lines and in the murine mammary gland during development. The expression of each of these kinases was analyzed throughout postnatal mammary gland development as well as in a panel of mammary epithelial cell lines derived from distinct transgenic models of breast cancer. Although the majority of protein kinases isolated in this screen have no currently recognized role in mammary development, most kinases examined were found to exhibit developmental regulation. After kinases were clustered on the basis of similarities in their temporal expression profiles during mammary development, multiple distinct patterns of expression were observed. Analysis of these patterns revealed an ordered set of expression profiles in which successive waves of kinase expression occur during development. Interestingly, several protein kinases whose expression has previously been reported to be restricted to tissues other than the mammary gland were isolated in this screen and found to be expressed in the mammary gland. In aggregate, these findings suggest that the array of kinases participating in the regulation of normal mammary development is considerably broader than currently appreciated.
Assuntos
Glândulas Mamárias Animais/enzimologia , Glândulas Mamárias Animais/crescimento & desenvolvimento , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Divisão Celular , Linhagem Celular , Primers do DNA/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , Lactação/genética , Lactação/metabolismo , Glândulas Mamárias Animais/citologia , Camundongos , Dados de Sequência Molecular , Gravidez , Proteínas Quinases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The steroid hormones 17 beta-estradiol and progesterone play a central role in the pathogenesis of breast cancer and regulate key phases of mammary gland development. This suggests that developmental regulatory molecules whose activity is influenced by ovarian hormones may also contribute to mammary carcinogenesis. In a screen designed to identify protein kinases expressed in the mammary gland, we previously identified a novel SNF1-related serine/threonine kinase, Hunk (hormonally upregulated Neu-associated kinase). During postnatal mammary development, Hunk mRNA expression is restricted to a subset of mammary epithelial cells and is temporally regulated with highest levels of expression occurring during early pregnancy. In addition, treatment of mice with 17 beta-estradiol and progesterone results in the rapid and synergistic upregulation of Hunk expression in a subset of mammary epithelial cells, suggesting that the expression of this kinase may be regulated by ovarian hormones. Consistent with the tightly regulated pattern of Hunk expression during pregnancy, mammary glands from transgenic mice engineered to misexpress Hunk in the mammary epithelium manifest temporally distinct defects in epithelial proliferation and differentiation during pregnancy, and fail to undergo normal lobuloalveolar development. Together, these observations suggest that Hunk may contribute to changes in the mammary gland that occur during pregnancy in response to ovarian hormones.
Assuntos
Glândulas Mamárias Animais/fisiologia , Prenhez/fisiologia , Proteínas Serina-Treonina Quinases/genética , Animais , Diferenciação Celular , Células Epiteliais/citologia , Estradiol/farmacologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Lactoferrina/genética , Glândulas Mamárias Animais/anatomia & histologia , Vírus do Tumor Mamário do Camundongo , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Ovário/fisiologia , Gravidez , Progesterona/farmacologia , Sequências Repetidas TerminaisRESUMO
We previously identified a novel protein kinase, Hunk, by means of a degenerate PCR screen designed to isolate kinases expressed in the murine mammary gland. We now describe the molecular cloning, chromosomal localization, and activity of this kinase and characterize its spatial and temporal pattern of expression in the mouse. We have isolated a 5.0-kb full-length cDNA clone that contains the 714-amino-acid open reading frame encoding Hunk. Analysis of this cDNA reveals that Hunk is most closely related to the SNF1 family of serine/threonine kinases and contains a newly described SNF1 homology domain. Accordingly, antisera specific for Hunk detect an 80-kDa polypeptide with associated phosphotransferase activity. Hunk is located on distal mouse chromosome 16 in a region of conserved synteny with human chromosome 21q22. During fetal development and in the adult mouse, Hunk mRNA expression is developmentally regulated and tissue-specific. Moreover, in situ hybridization analysis reveals that Hunk expression is restricted to subsets of cells within a variety of organs in the adult mouse. These findings suggest a role for Hunk in murine development.
Assuntos
Proteínas Serina-Treonina Quinases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Linhagem Celular , Clonagem Molecular , Embrião de Mamíferos/enzimologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Immunoblotting , Hibridização In Situ , Glândulas Mamárias Animais/enzimologia , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Especificidade de Órgãos , Reação em Cadeia da Polimerase , Proteínas Serina-Treonina Quinases/isolamento & purificação , Proteínas Serina-Treonina Quinases/metabolismo , Alinhamento de SequênciaRESUMO
Calcium is an important second messenger in eukaryotic cells. Many of the effects of calcium are mediated via its interaction with calmodulin and the subsequent activation of Ca(2+)/calmodulin-dependent (CaM) kinases. CaM kinases are involved in a wide variety of cellular processes including muscle contraction, neurotransmitter release, cell cycle control, and transcriptional regulation. While CaMKII has been implicated in learning and memory, the biological role of the other multifunctional CaM kinases, CaMKI and CaMKIV, is largely unknown. In the course of a degenerate RT-PCR protein kinase screen, we identified a novel serine/threonine kinase, Pnck. In this report, we describe the cloning, chromosomal localization, and expression of Pnck, which encodes a 38-kDa protein kinase whose catalytic domain shares 45-70% identity with members of the CaM kinase family. The gene for Pnck localizes to mouse chromosome X, in a region of conserved synteny with human chromosome Xq28 that is associated with multiple distinct mental retardation syndromes. Pnck is upregulated during intermediate and late stages of murine fetal development with highest levels of expression in developing brain, bone, and gut. Pnck is also expressed in a tissue-specific manner in adult mice with highest levels of expression detected in brain, uterus, ovary, and testis. Interestingly, Pnck expression in these tissues is restricted to particular compartments and appears to be further restricted to subsets of cells within those compartments. The chromosomal localization of Pnck, along with its tissue-specific and restricted pattern of spatial expression during development, suggests that Pnck may be involved in a variety of developmental processes including development of the central nervous system.