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The αvβ6 integrin is up-regulated on epithelial malignancies and has been implicated in various aspects of cancer progression. Immunohistochemical analysis of αvβ6 expression in 10 human tumor types showed increased expression relative to normal tissues. Squamous carcinomas of the cervix, skin, esophagus, and head and neck exhibited the highest frequency of expression, with positive immunostaining in 92% (n = 46), 84% (n = 49), 68% (n = 56), and 64% (n = 100) of cases, respectively. We studied the role of αvβ6 in Detroit 562 human pharyngeal carcinoma cells in vitro and in vivo. Prominent αvβ6 expression was detected on tumor xenografts at the tumor-stroma interface resembling the expression on human head and neck carcinomas. Nonetheless, coculturing cells in vitro with matrix proteins did not up-regulate αvβ6 expression. Detroit 562 cells showed αvβ6-dependent adhesion and activation of transforming growth factor-β (TGF-β) that was inhibited >90% with an αvβ6 blocking antibody, 6.3G9. Although both recombinant soluble TGF-β receptor type-II (rsTGF-βRII-Fc) and 6.3G9 inhibited TGF-β–mediated Smad2/3 phosphorylation in vitro, there was no effect on proliferation. Conversely, in vivo, 6.3G9 and rsTGF-βRII-Fc inhibited xenograft tumor growth by 50% (n = 10, P < 0.05) and >90% (n = 10, P < 0.001), respectively, suggesting a role for the microenvironment in this response. However, stromal collagen and smooth muscle actin content in xenograft sections were unchanged with treatments. Although further studies are required to consolidate in vitro and in vivo results and define the mechanisms of tumor inhibition by αvβ6 antibodies, our findings support a role for αvβ6 in human cancer and underscore the therapeutic potential of function blocking αvβ6 antibodies. [Cancer Res 2008;68(2):561–70]

We have generated mutant mouse estrogen receptors which differ in their sensitivity to estrogen and the antiestrogen 4-hydroxytamoxifen. Mutation of the glycine at position 525 and the methionine and/or serine at positions 521/522 virtually abolishes the ability of the receptor to bind estradiol and stimulate transcription. In contrast, the mutant receptors retain the partial agonist activity exhibited by the wild-type receptor in the presence of 4-hydroxytamoxifen. The mutations do not affect the expression and DNA-binding activity of the receptor, but do abolish the estrogen-induced increase in the mobility of the receptor-DNA complex observed with the wild-type receptor. Other mutant receptors that were able to bind and stimulate transcription in the presence of estradiol also failed to show the agonist-induced increase in the mobility of the receptor-DNA complex, suggesting that it is unlikely to reflect the formation of a hormone-dependent transcriptional activation function.

We have investigated the effects of ligand and DNA binding on the structure of the oestrogen receptor by performing limited proteolysis and analysing DNA binding activity by gel shift analysis. The effects of oestradiol, 4-hydroxytamoxifen and ICI 164,384 have been examined and we have found that despite differences in the DNA binding activity or relative mobility of the receptor-DNA complex we were unable to detect differences in the cleavage pattern produced by trypsin, chymotrypsin, Staphylococcus aureus V8, papain or elastase. Inhibition of DNA binding by ICI 164,384 was lost in receptor fragments that lacked the hormone binding domain. In contrast to the full-length receptor, proteolytic fragments produced by chymotrypsin differed in their ability to bind to an oestrogen response element (ERE) vs a thyroid response element (TRE). Evidence is presented that this difference can be accounted for by the inability of fragments lacking the hormone binding domain to dimerise on a TRE.

Sequences essential for dimerisation have been identified in the hormone binding domain of the mouse oestrogen receptor by insertional and point mutagenesis and sequence comparisons reveal that equivalent residues may be conserved in other members of the nuclear hormone receptor superfamily. To assess functional compatibility of this region between members of the receptor superfamily, peptide sequences corresponding to the equivalent regions of the human androgen receptor and retinoic acid receptor have been substituted for the dimerisation domain of the mouse oestrogen receptor. The resulting chimeric proteins were analysed for high affinity DNA binding using a gel retardation assay and shown to bind with reduced affinity compared to the wild type oestrogen receptor. The reduction in DNA binding observed may result from the intramolecular incompatibility of functional elements within the hormone binding domain of nuclear hormone receptors.

We have identified two transactivation domains in the mouse oestrogen receptor whose activities depend on the target promoter. The major domain is contained within the C-terminal portion of the protein and depends upon oestrogen binding for its activity. The location and oestrogen dependence of this domain has been confirmed using chimaeric receptors containing the Lex A DNA binding domain. Although transactivation by the C-terminal domain is dependent upon ligand binding the analysis of receptor deletion mutants has demonstrated that these two functions are not entirely coincident. The second transactivation domain lies within the N-terminal region and is active in the absence of oestradiol. The differences in oestrogen requirement for the activity of the two transactivation domains may account for the partial agonist activity of certain antihormones.

The seminal vesicles of the rat synthesise large amounts of androgen-regulated secretory proteins. Indirect immunofluorescence cytochemistry and immunoblotting with monospecific polyclonal antibodies against three of the major secretory proteins (II, S and F) have been used to investigate the tissue distribution, subcellular localisation, androgen-regulation and developmental profile of secretory protein synthesis. There was no evidence for regional specialisation of the seminal vesicle epithelium; every epithelial cell synthesizes all three proteins via a classical secretory involving storage in secretory vesicles. Proteins S and II are contained within the same secretory vesicles. The time course of deinduction of proteins S and F after castration and their reinduction by testosterone closely followed that for their specific mRNAs described previously. During development, proteins S and F first appear between 10 and 15 days after birth. A protein immunologically related to seminal vesicle protein II is present in the lateral and dorsal lobes of the prostatic complex.

The seminal vesicles of male rat secrete tissue-specific proteins under androgenic control. The effects of testosterone on the rough endoplasmic reticulum (RER) and Golgi-system of this tissue have been quantified using specific antibodies. Castration was followed within 2 weeks by a 10-fold reduction in RER-specific membrane protein. This was reversed by testosterone commencing about 4 h after exposure to the hormone. Five individual major RER antigens were separately quantified; these changed coordinately in response to androgen. No hormone-induced changes were seen in Golgi-specific membrane protein. Hormonal effects on mRNAs for two major secretory proteins were also measured using hybridisation to specific cDNA probes. The cellular concentrations of the two mRNAs changed by at least 1000-fold during hormonal treatment. A detailed examination of the time-course of induction by testosterone failed to show any temporal distinction between effects on mRNA and RER.

Addition of mammalian insulin to a nutritionally rich, chemically defined culture medium affects Neurospora crassa "slime" (wall-less) cells, as indicated by enhancement of growth, extension of viability at the stationary phase of growth, alteration of morphology, and stimulation of glucose oxidation. Bovine, porcine, and recombinant human insulin had similar effects on growth and morphology, while proinsulin, reduced insulin, and several other proteins were inactive. Insulin added in the presence of excess antiinsulin antibody was without activity. Intact cells possessed high affinity insulin-binding sites, represented by a curvilinear Scatchard plot, suggesting that effects are mediated through insulin receptors on the cell surface. These findings establish a role for insulin or insulin-like molecules in regulating growth and metabolism in this fungal cell and demonstrate a close similarity to insulin effects on certain mammalian cells.

A consensus sequence has been proposed as necessary for steroid binding on the basis of sequence homology in steroidogenic enzymes, steroid binding proteins and steroid receptors. We have mapped the limit of sequences important for steroid binding in the mouse estrogen receptor using Scatchard analysis of deletion mutants. Since a mutant lacking the entire proposed consensus is still able to bind estradiol with essentially wild type affinity, the functional significance of this motif is unclear.

All the major androgen-regulated secretory proteins of rat seminal vesicles have been purified in high yield from polyacrylamide gels using electroelution. In the process a sixth previously undocumented protein has been identified. Amino acid compositions of all the proteins are very similar and highly unusual, being high in lysine and arginine, and with 40-50% of the residues accounted for by serine, glycine and glutamate/glutamine. N-Terminal amino acid sequences for 3 of the proteins show that they are clearly the products of related genes. At least one of the other proteins is N-terminally blocked in vivo. Antibodies specific for each protein have been raised and provide evidence of structural similarity between the proteins. The antibodies were also used in immunofluorescence histochemistry with the rat copulatory plug, showing for the first time that all the major proteins of seminal vesicle secretion are components of this reproductive structure.

An in vitro system has been used to study the role of purified rat seminal vesicle proteins in the formation of the copulatory vaginal plug. Proteins II, IV (or S) and V (or F) were each separately coagulated using the transglutaminase in coagulating gland extracts. In each case the coagulum required Ca2+ ions for its formation and was insoluble in denaturing solvents. In experiments with [3H]lysine, proteins II and S incorporated [3H]lysine into glu-lys dipeptide with similar kinetics. Both the N-terminal and C-terminal glutamine residues of protein S participated in the reaction.

Addition of bovine insulin to cells of the wallless variant FGSC4761 of Neurospora crassa (“slime”) produced several significant effects on glycogen metabolism. 1) Intracellular levels of the glycogen precursor UDP-glucose decreased 17–18% (P < 0.01) within 30 min of insulin addition. 2) Cells grown with insulin possessed 40% more glycogen than did control cells. 3) The incorporation of 14C-labeled glucose into glycogen increased 41% after 30-min treatment with 100 nM bovine insulin (P < 0.01). 4) Insulin treatment of the cells caused activation of the enzyme glycogen synthase from a glucose-6- phosphate-dependent form to an independent form. Half-maximum activation occurred with 2 nM insulin. These are similar to insulin-induced effects in some mammalian cells. In contrast, no insulin-induced effect on glucose transport could be demonstrated in these cells. (Endocrinology 122: 518–523, 1988)

The copulatory vaginal plug is a conspicuous feature of rodent reproduction. The five major seminal vesicle secretory proteins of Rattus norvegicus (proteins I-V), which form the copulatory plug, constitute a closely related androgen-regulated family that appears to share a common evolutionary origin. The relationships between these rat proteins and the major seminal vesicle proteins of other rodents were explored using antibodies specific for the individual rat proteins. Immunoblotting of proteins separated by SDS-PAGE showed that the vesicular proteins of R. rattus are identical to those of R. norvegicus except for an additional protein related to protein III. No differences were seen in inbred and outbred strains of R. norvegicus. Of the major proteins of Mus musculus, one showed strong homology with rat protein II and three others were weakly homologous to proteins I, IV (or S) and V (or F); none showed homology to rat protein III. The only homology between the vesicular proteins of Mesocricetus auratus (Syrian hamster) and Meriones ungulatus (Mongolian gerbil) was with rat protein II while those of Cavia porcellus (guinea pig) showed no homology at all with the rat proteins. In addition, cDNA probes for rat genes IV and V both detected weak homologues in seminal vesicle RNA from mice but not guinea pigs.(ABSTRACT TRUNCATED AT 250 WORDS)