Involution of the mouse mammary gland is associated with an immune cascade and an acute-phase response, involving LBP, CD14 and STAT3

INTRODUCTION: Involution of the mammary gland is a complex process of controlled apoptosis and tissue remodelling. The aim of the project was to identify genes that are specifically involved in this process. METHODS: We used Affymetrix oligonucleotide microarrays to perform a detailed transcript analysis on the mechanism of controlled involution after withdrawal of the pups at day seven of lactation. Some of the results were confirmed by semi-quantitative reverse transcriptase polymerase chain reaction, Western blotting or immunohistochemistry. RESULTS: We identified 145 genes that were specifically upregulated during the first 4 days of involution; of these, 49 encoded immunoglobulin genes. A further 12 genes, including those encoding the signal transducer and activator of transcription 3 (STAT3), the lipopolysaccharide receptor (CD14) and lipopolysaccharide-binding protein (LBP), were involved in the acute-phase response, demonstrating that the expression of acute-phase response genes can occur in the mammary gland itself and not only in the liver. Expression of LBP and CD14 was upregulated, at both the RNA and protein level, immediately after pup withdrawal; CD14 was strongly expressed in the luminal epithelial cells. Other genes identified suggested neutrophil activation early in involution, followed by macrophage activation late in the process. Immunohistochemistry and histological staining confirmed the infiltration of the involuting mammary tissue with neutrophils, plasma cells, macrophages and eosinophils. CONCLUSION: Oligonucleotide microarrays are a useful tool for identifying genes that are involved in the complex developmental process of mammary gland involution. The genes identified are consistent with an immune cascade, with an early acute-phase response that occurs in the mammary gland itself and resembles a wound healing process

Mutations of the BRAF gene in human cancer

Cancers arise owing to the accumulation of mutations in critical genes that alter normal programmes of cell proliferation, differentiation and death. As the first stage of a systematic genome-wide screen for these genes, we have prioritized for analysis signalling pathways in which at least one gene is mutated in human cancer. The RAS RAF MEK ERK MAP kinase pathway mediates cellular responses to growth signals. RAS is mutated to an oncogenic form in about 15% of human cancer. The three RAF genes code for cytoplasmic serine/threonine kinases that are regulated by binding RAS. Here we report BRAF somatic missense mutations in 66% of malignant melanomas and at lower frequency in a wide range of human cancers. All mutations are within the kinase domain, with a single substitution (V599E) accounting for 80%. Mutated BRAF proteins have elevated kinase activity and are transforming in NIH3T3 cells. Furthermore, RAS function is not required for the growth of cancer cell lines with the V599E mutation. As BRAF is a serine/threonine kinase that is commonly activated by somatic point mutation in human cancer, it may provide new therapeutic opportunities in malignant melanoma

p53 mutation with frequent novel codons but not a mutator phenotype in BRCA1- and BRCA2-associated breast tumours

The status of p53 was investigated in breast tumours arising in germ-line carriers of mutant alleles of BRCA1 and BRCA2 and in a control series of sporadic breast tumours. p53 expression was detected in 20/26 (77%) BRCA1-, 10/22 (45%) BRCA2-associated and 25/72 (35%) grade-matched sporadic tumours. Analysis of p53 sequence revealed that the gene was mutant in 33/50 (66%) BRCA-associated tumours, whereas 7/20 (35%) sporadic grade-matched tumours contained p53 mutation (P < 0.05). A number of the mutations detected in the BRCA-associated tumours have not been previously described in human cancer databases, whilst others occur extremely rarely. Analysis of additional genes, p16(INK4), Ki-ras and β-globin revealed absence or very low incidence of mutations, suggesting that the higher frequency of p53 mutation in the BRCA-associated tumours does not reflect a generalized increase in susceptibility to the acquisition of somatic mutation. Furthermore, absence of frameshift mutations in the polypurine tracts present in the coding sequence of the TGF β type II receptor (TGF β IIR) and Bax implies that loss of function of BRCA1 or BRCA2 does not confer a mutator phenotype such as that found in tumours with microsatellite instability (MSI). p21(Waf1) was expressed in BRCA-associated tumours regardless of p53 status and, furthermore, some tumours expressing wild-type p53 did not express detectable p21(Waf1). These data do not support, therefore, the simple model based on studies of BRCA-/- embryos, in which mutation of p53 in BRCA-associated tumours results in loss of p21(Waf1) expression and deregulated proliferation. Rather, they imply that proliferation of such tumours will be subject to multiple mechanisms of growth regulation

Human breast development

This review is intended to give an overview of current knowledge on human breast development. It focuses on the limitations of our understanding on the origins of human breast cancer in the context of this mainly morphological and static assessment of what is known about human breast development. The world literature is very limited and caution is needed in drawing analogies with the mouse. There is an increasing emphasis on research to understand normal stem cells in the breast on the assumption that these are the targets for the initiation of breast cancer. It is thus a priority to understand normal human breast development, but there are major obstacles to such studies mainly due to ethical considerations and to tissue acquisition

Development of novel selective cell ablation in the mammary gland and to study cell-cell interactions and chemoprevention

We have generated transgenic mice which express the gene encoding Escherichia coli nitroreductase (NTR) specifically in the luminal epithelial cells of the mammary gland and the glial cells of the brain. The enzyme activates an antitumour drug CB1954, to produce a cross-linking agent that kills all cells expressing the enzyme. We have shown that administration of the antitumour drug CB 1954 rapidly and selectively kills these cells. Original experiments demonstrated the ability to ablate the luminal cells in the mammary gland with no apparent bystander effect. Subsequently, astrocytes expressing nitroreductase under the targeting of the GFAP promoter were selectively ablated following administration of the prodrug CB 1954 produces a degeneration of granular neurones due to changes in glutamate levels. Recent experiments demonstrated inhibition of myc-dependent mammary tumours using the same enzyme (nitroreductase) - prodrug (CB 1954), combination. Owing to the ease of control of NTR-mediated cell ablation, we anticipate that this system will supersede herpes simplex virus type 1 thymidine kinase. There are widespread potential applications for this approach in the dissection of complex cellular interactions during development and in the adult organism. The present transgenic models also have important applications for the study in vivo of novel prodrugs that can be selected for variable degrees of bystander effects. Such studies will have particular significance for those groups advocating the use of NTR as an appropriate enzyme for gene-directed enzyme prodrug therapy by providing models of a wide range of human disease for mechanistic and therapeutic experimentation. The results clearly demonstrate that the model has potential to study chemoprevention and fundamental questions on cell-cell interactions in cell biolog

Development of novel selective cell ablation in the mammary gland and to study cell-cell interactions and chemoprevention

We have generated transgenic mice which express the gene encoding Escherichia coli nitroreductase (NTR) specifically in the luminal epithelial cells of the mammary gland and the glial cells of the brain. The enzyme activates an antitumour drug CB1954, to produce a cross-linking agent that kills all cells expressing the enzyme. We have shown that administration of the antitumour drug CB 1954 rapidly and selectively kills these cells. Original experiments demonstrated the ability to ablate the luminal cells in the mammary gland with no apparent bystander effect. Subsequently, astrocytes expressing nitroreductase under the targeting of the GFAP promoter were selectively ablated following administration of the prodrug CB 1954 produces a degeneration of granular neurones due to changes in glutamate levels. Recent experiments demonstrated inhibition of myc-dependent mammary tumours using the same enzyme (nitroreductase) - prodrug (CB 1954), combination. Owing to the ease of control of NTR-mediated cell ablation, we anticipate that this system will supersede herpes simplex virus type 1 thymidine kinase. There are widespread potential applications for this approach in the dissection of complex cellular interactions during development and in the adult organism. The present transgenic models also have important applications for the study in vivo of novel prodrugs that can be selected for variable degrees of bystander effects. Such studies will have particular significance for those groups advocating the use of NTR as an appropriate enzyme for gene-directed enzyme prodrug therapy by providing models of a wide range of human disease for mechanistic and therapeutic experimentation. The results clearly demonstrate that the model has potential to study chemoprevention and fundamental questions on cell-cell interactions in cell biolog

Tissue banks and cell lines derived from patients with a high risk of breast cancer and in situ disease

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