Phosphorylation near nuclear localization signal regulates nuclear import of adenomatous polyposis coli protein.
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Mutation of the adenomatous polyposis coli (APC) gene is an early step in the development of colorectal carcinomas. APC protein is located in both the cytoplasm and the nucleus. The objective of this study was to define the nuclear localization signals (NLSs) in APC protein. APC contains two potential NLSs comprising amino acids 1767-1772 (NLS1(APC)) and 2048-2053 (NLS2(APC)). Both APC NLSs are well conserved among human, mouse, rat, and fly. NLS1(APC) and NLS2(APC) each were sufficient to target the cytoplasmic protein beta-galactosidase to the nucleus. Mutational analysis of APC demonstrated that both NLSs were necessary for optimal nuclear import of full-length APC protein. Alignment of NLS2(APC) with the simian virus 40 large T antigen NLS (NLS(SV40 T-ag)) revealed sequence similarity extending to adjacent phosphorylation sites. Changing a serine residue (Ser(2054)) to aspartic acid mutated the potential protein kinase A site adjacent to NLS2(APC), resulting in both inhibition of the NLS2(APC)-mediated nuclear import of a chimeric beta-galactosidase fusion protein and a reduction of full-length APC nuclear localization. Our data provide evidence that control of APC's nuclear import through phosphorylation is a potential mechanism for regulating APC's nuclear activity. (+info)
A novel cell culture model for studying differentiation and apoptosis in the mouse mammary gland.
(42/1118)
BACKGROUND: This paper describes the derivation and characterization of a novel, conditionally immortal mammary epithelial cell line named KIM-2. These cells were derived from mid-pregnant mammary glands of a mouse harbouring one to two copies of a transgene comprised of the ovine beta-lactoglobulin milk protein gene promoter, driving expression of a temperature-sensitive variant of simian virus-40 (SV40) large T antigen (T-Ag). RESULTS: KIM-2 cells have a characteristic luminal epithelial cell morphology and a stable, nontransformed phenotype at the semipermissive temperature of 37 degrees C. In contrast, at the permissive temperature of 33 degrees C the cells have an elongated spindle-like morphology and become transformed after prolonged culture. Differentiation of KIM-2 cells at 37 degrees C, in response to lactogenic hormones, results in the formation of polarized dome-like structures with tight junctions. This is accompanied by expression of the milk protein genes that encode beta-casein and whey acidic protein (WAP), and activation of the prolactin signalling molecule, signal transducer and activator of transcription (STAT)5. Fully differentiated KIM-2 cultures at 37 degrees C become dependent on lactogenic hormones for survival and undergo extensive apoptosis upon hormone withdrawal, as indicated by nuclear morphology and flow cytometric analysis. KIM-2 cells can be genetically modified by stable transfection and clonal lines isolated that retain the characteristics of untransfected cells. CONCLUSION: KIM-2 cells are a valuable addition, therefore, to currently available lines of mammary epithelial cells. Their capacity for extensive differentiation in the absence of exogenously added basement membrane, and ability to undergo apoptosis in response to physiological signals will provide an invaluable model system for the study of signal transduction pathways and transcriptional regulatory mechanisms that control differentiation and involution in the mammary gland. (+info)
Reduced mammary tumor progression in WAP-TAg/WAP-maspin bitransgenic mice.
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Maspin is a unique serpin involved in the suppression of tumor growth and metastasis. To investigate whether increased levels of maspin protect against tumor progression in vivo, we established a transgenic model in which maspin is targeted to mammary epithelial cells by the Whey Acidic Protein (WAP) promoter for overexpression. We crossed these WAP-maspin transgenic mice with the WAP-TAg mouse model of tumor progression. Maspin overexpression increased the rate of apoptosis of both preneoplastic and carcinomatous mammary epithelial cells. Maspin reduced tumor growth through a combination of reduced angiogenesis and increased apoptosis. The number of pulmonary metastases was reduced in the presence of maspin overexpression. These data demonstrate that targeted overexpression of maspin can inhibit tumor progression in vivo, likely through a combination of increased apoptosis, decreased angiogenesis, and inhibition of tumor cell migration. (+info)
Estrogen promotes mammary tumor development in C3(1)/SV40 large T-antigen transgenic mice: paradoxical loss of estrogen receptoralpha expression during tumor progression.
(44/1118)
Although several lines of epidemiological evidence suggest that estrogen exposure influences the incidence of breast cancer development, the mechanisms by which estrogen may stimulate the formation of breast cancer remain poorly understood. We have explored how alterations in estrogen exposure can influence the development of mammary cancer in the C3(1)/T(AG) transgenic model, where estrogen levels and estrogen receptor alpha (ERalpha) expression do not appear to modify the level of transgene expression. The C3(1)/T(AG) transgene becomes transcriptionally active in mammary ductal target cells at 3 weeks of age after the estrogen-induced differentiation of the mammary epithelial anlage to the ductal outgrowth stage. Complete maturation of the mammary ductal tree, however, is not required for cancer development because tumors arise in animals where ductal branching and terminal end bud formation have been prematurely arrested by ovariectomy. Mammary tumorigenesis in this model is promoted by increased estrogen exposure with the development of significantly more mammary intraepithelial neoplastic lesions and carcinomas associated with accelerated malignant conversion. The promotion of mammary tumors in this model appears to occur through an estrogen-induced proliferation and increase in the number of available target cells for transformation at the terminal ductal lobular units, as has been postulated to occur in women who receive hormone replacement therapy and/or by additional molecular mechanisms. We show, for the first time in a transgenic mouse model, that mammary tumor progression is associated with the loss of ERalpha expression, as has been often observed in human breast cancers with important clinical significance. Estrogen signaling may, therefore, serve different functions, depending upon the stage of tumorigenesis. ERbeta expression is up-regulated during tumor progression, although the functional significance of this remains to be determined. (+info)
Effect on polyomavirus T-antigen function of mutations in a conserved leucine-rich segment of the DnaJ domain.
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The N-terminal part of the mouse polyomavirus T antigens contains a highly conserved segment (-LLELLKL-), including amino acid residues 13 to 19. The sequence motif is predicted to form alpha helix I in the DnaJ domain of the T antigens. Four mutants with conservative substitutions of amino acid residues 13 and 14 were constructed. Of the four substitutions, L13M, L13I, L13V, and L14V, only L13V resulted in a phenotypic change. In transfected mouse cells, L13V large T antigen showed a more than 100-fold-reduced viral DNA synthesis. The viral replication could not be rescued by cotransfection of the cells with DNA expressing small t antigen or a large T antigen truncated at the C terminus that would compensate for a defect in host cell stimulation. In contrast to the effect on DNA replication, the L13V substitution in large T antigen did not prevent complex formation with Hsc70 and the Rb protein. Also, the activity of the protein in transactivation of transcription from the adenovirus E2 promoter was unimpaired, showing that the transcription factor E2F was released from pRb. The L13V substitution also caused a defect in small t antigen. However, this phenotypic change was due to protein instability. In contrast, middle T antigen with the L13V substitution remained stable and functional in cellular transformation. Together, the data show that the effect of the L13V substitution did not abrogate the Hsc70 interaction of the DnaJ domain. However, it is possible that the substitution of amino acid residue 13 affected specific DnaJ functions of large T antigen. (+info)
Regulation of nuclear import by light-induced activation of caged nuclear localization signal in living cells.
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A novel fluorescence probe suitable for the study of nuclear import in living cells has been developed. The lysine-128 residue in SV40 T-antigen nuclear localization signal (NLS) was converted to a caged lysine with the amino acid blocked by a photocleavable protecting group. Following irradiation of ultraviolet (UV) light, the caged NLS conjugate translocated into and accumulated in the nucleus within 20 min similar to uncaged NLS conjugate. Maximum import rate saturated approximately 4.78+/-0.21% per minute when the duration of irradiation was more than 1/15 s (22 mW/cm(2)). Caged NLS conjugate tended to distribute near the surface of the nucleus, and this association became stronger after UV irradiation. The caged conjugate enabled us to regulate the initial state of the reaction, both spatially and temporally. (+info)
Cytotoxic T lymphocytes from HLA-A2.1 transgenic mice define a potential human epitope from simian virus 40 large T antigen.
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Recent reports have documented the presence of SV40 large T antigen (T ag) sequences in a number of human tumors and raised the question of whether cellular immunity to T ag is elicited in such individuals. We used HLA-A2.1 transgenic C57BL/6 mice to identify an epitope from T ag recognized by CD8+ CTLs when presented by this human MHC class I molecule. Immunization of HLA-A2.1 transgenic mice with syngeneic T ag-transformed cells resulted in the induction of HLA-A2.1-restricted, T ag-specific CTLs. The target epitope, residues 281-289 (KCDDVLLLL) of T ag, was identified using both cell lines expressing T ag variants and synthetic T ag peptides. Peptide 281-289 bound stably to HLA-A2.1 molecules, effectively sensitized target cells for CTL lysis, and was efficiently processed from endogenous T ag in cells of both mouse and human origin. CTLs were not cross-reactive on the human BK or JC virus T ags. Thus, SV40 T ag 281-289 represents a potential specific CTL recognition epitope for humans. (+info)
High levels of ceruloplasmin in the serum of transgenic mice developing hepatocellular carcinoma.
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Transgenic mice expressing the Simian virus 40 large T antigen under the control of the liver-specific human antithrombin-III promoter all develop well-differentiated hepatocellular carcinoma. During tumour development serum ceruloplasmin (Cp) increases gradually until it reaches 30 times control levels in all transgenic mice at 6 months of age. The accumulation of Cp in the serum is due to the increased transcription of the Cp gene as well as to the increase in Cp mRNA stability in the livers of the transgenic mice. One-half of the overproduced Cp is charged with copper and Cp-associated serum oxidase activity increases in parallel with the holo-Cp concentration. Through its ferroxidase activity Cp is involved prominently in iron metabolism. Analysis of copper and iron in serum and liver revealed increased copper levels in the serum of tumour-bearing animals and which increased in parallel with Cp concentration; the amounts of copper in the liver were unchanged. In contrast, serum iron remained constant during tumour development whereas the iron concentration in the livers of the transgenic mice decreased. (+info)