Identification of heat shock protein 10 within the equine embryo, endometrium, and maternal peripheral blood mononuclear cells.

Early pregnancy factor has been identified as a 10-kDa extracellular homolog of heat shock protein 10 (Hsp10). Hsp10 has been detected during early pregnancy in serum of mice, sheep, pigs, horses, cows, and humans by the rosette inhibition test. Hsp10 has also been associated with several neoplastic and autoimmune diseases. The goal of the present study was to determine if Hsp10 could be detected in the early equine embryo through the use of immunohistochemistry and quantitative real-time PCR. Additionally, analysis of systemically harvested peripheral blood mononuclear cells (PBMCs) from both pregnant and nonpregnant mares was evaluated to determine expression levels of HSP10. Embryos were collected from Quarter Horse mares by uterine lavage at either 8 or 25 days after ovulation. Collection and separation of PBMCs occurred on Day 8 for both pregnant and nonpregnant mares. Immunohistochemistry revealed cytoplasmic localization of HSP10 throughout the single layer of ectodermal cells forming the trophoblast in Day-8 embryos. Day-25 embryos demonstrated intense localization focally along the apical border of ectodermal cells forming the trophoblast layer of the developing chorion. There was no nuclear staining in either embryonic population. Quantitative real-time PCR detected the presence of mRNA for HSP10 in both 8- and 25-day equine embryos. Day-25 embryos exhibited an elevated degree of expression (P = 0.006) compared with the 8-day embryos for HSP10. Endometrial samples did not display any significant difference in degree of expression for HSP10 (P = 0.10). Finally, PBMCs from pregnant mares demonstrated elevated (P = 0.03) expression of HSP10 compared to the nonpregnant mares on Day 8 of the estrous cycle. This study confirmed the presence of HSP10 protein and mRNA expression of HSP10 in equine embryos at two maturation stages. Additionally, the presence of increased gene expression within PBMCs of pregnant mares suggests communication, possibly leading to necessary immunomodulatory effects between the embryo and mare.

EGFR regulation by microRNA in lung cancer: correlation with clinical response and survival to gefitinib and EGFR expression in cell lines.

BACKGROUND: Allelic loss in chromosome 3p is one of the most frequent and earliest genetic events in lung carcinogenesis. We investigated if the loss of microRNA-128b, a microRNA located on chromosome 3p and a putative regulator of epidermal growth factor receptor (EGFR), correlated with response to targeted EGFR inhibition. Loss of microRNA-128b would be equivalent to losing a tumor suppressor gene because it would allow increased expression of EGFR. PATIENTS AND METHODS: We initially showed that microRNA-128b is a regulator of EGFR in non-small-cell lung cancer (NSCLC) cell lines. We tested microRNA-128b expression levels by quantitative RT-PCR, genomic copy number by quantitative PCR, and mutations in the mature microRNA-128b by sequencing. We determined whether microRNA-128b loss of heterozygosity (LOH) in 58 NSCLC patient samples correlated with response to gefitinib and evaluated EGFR expression and mutation status. RESULTS: We determined that microRNA-128b directly regulates EGFR. MicroRNA-128b LOH was frequent in tumor samples and correlated significantly with clinical response and survival following gefitinib. EGFR expression and mutation status did not correlate with survival outcome. CONCLUSION: Identifying microRNA regulators of oncogenes could have far-reaching implications for lung cancer patients including improving patient selection for targeted agents, development of novel therapeutics, or development as early biomarkers of disease.

Native American cancer education: genetic and cultural issues.

BACKGROUND: The authors met with intertribal groups to learn about cultural issues related to cancer genetics. The information gathered from these meetings identified issues that are incorporated into the Genetic Education for Native Americans (GENA) interactive, innovative and multidisciplinary curriculum. METHODS: To address the diverse cultural and scientific issues, the faculty presented customized workshops during conferences for Native American college students. RESULTS: The authors discuss current issues and techniques in cancer education in Native American communities. CONCLUSIONS: Better understanding of tribal culture among researchers will enhance Native Americans' collaboration in research.

Reproductive state of rat mammary gland stroma modulates human breast cancer cell migration and invasion.

It has been established that the invasive behavior of cancer cells can be regulated by alterations in their extracellular environment. We investigated whether extracellular matrix isolated from nulliparous and postlactating (involuting) rat mammary glands differentially modulated the metastatic behavior of human breast cancer cells. Using modified Boyden chamber and three-dimensional culture assays, nulliparous mammary matrix was found to suppress motility and invasion in highly metastatic MDA-MB-435 cells, whereas involution mammary matrix supported motility and invasion in highly metastatic MDA-MB-435 cells, but not in cells with low metastatic potential. Biochemical characterization of the matrices revealed intact fibronectin (FN) and low matrix metalloproteinase activity in nulliparous mammary matrix and fragmented FN and high matrix metalloproteinase activity in the matrix isolated from involuting glands. Purified intact FN was found to inhibit cell invasiveness, whereas FN fragments enhanced cell invasiveness in a matrix metalloproteinase-dependent manner. These data suggest that physiological changes that occur in the mammary extracellular matrix as a result of reproductive status alter the in vitro parameters of metastatic potential.

Use of the yeast two-hybrid system for identifying the cascade of protein interactions resulting in apoptotic cell death.

Use of the yeast two-hybrid system allows rapid identification of interacting protein or proteins for a specific target protein. The technique is readily applied and allows immediate isolation of a cDNA encoding the interacting protein. One consideration might be to outline criteria for continued study of the interactors once they are identified. Our criterion for further study of an interactor is its presence in the mammary gland at a developmental time when the target protein is also present. Further characterization of interactors may involve immunoprecipitation, enzyme assays, or other techniques applicable to the specific protein.

Programmed cell death during mammary gland involution.

Understanding the cascade of gene expression and subsequent protein interactions that result both in the death of secretory mammary epithelium and the remodeling and renewal of the mammary gland for another cycle of lactation poses significant challenges (see Chapters 7 and 8, this volume). The complexity of mammary gland involution warrants caution in sorting through the various potential regulators and executors of apoptotic cell death in the mammary gland. As demonstrated by the number of remodeling enzymes expressed during involution, the relationship between mammary epithelium and its related mesenchyme is important for maintenance of differentiated function (Barcellos-Hoff et al., 1989; Streuli et al., 1991). Components of the extracellular matrix may play the role of survival factors, or may provide a source of factors, as a reserve of matrix-bound growth factors, necessary for survival of the secretory epithelium. Perturbation of this interaction alters mammary-specific differentiation gene expression, for example, production of milk proteins (Parry et al., 1987; Strange et al., 1991; Talhouk et al., 1992). Thus, alteration of the interaction between epithelium and its associated mesenchyme, which is an integral part of mammary involution, may also play a role in epithelial cell death. However, the epithelial-mesenchymal interactions that are the determining features in either mediating or modulating this cell death are just beginning to be defined. Stimuli that alter differentiated function may also induce apoptotic cell death of the epithelium but may have no physiological correlate. They may, however, have significant application in prevention or control of breast neoplasia.

Cloning, expression, and purification of a functional nonacetylated mammalian mitochondrial chaperonin 10.

An intact mouse mitochondrial chaperonin 10 has been cloned, sequenced, and overexpressed in Escherichia coli as a fusion protein harboring an oligohistidine tail at its COOH terminus. The latter was added to simplify protein purification. The purified protein is free of contaminating groES from the bacterial host cells. Edman degradation reveals that the initiator Met residue of the recombinant protein is removed in vivo, similar to the authentic chaperonin 10 purified from rat liver mitochondria. However, in contrast to the latter, the amino-terminal Ala residue of the recombinant protein is not acetylated; the molecular mass determined by electrospray ionization mass spectrometry is 12,350.9 +/- 2.6 daltons, in agreement with that predicted for the nonacetylated protein (12,351.2 daltons). Facilitated protein folding experiments with ribulose-biphosphate carboxylase, under "nonpermissive" in vitro conditions, demonstrate that the recombinant protein is fully functional with groEL. Thus, both the initial rates of protein folding and final yields observed with this heterologous combination are virtually identical to those obtained with groEL and groES. More important, like the authentic protein purified from mitochondria, the recombinant mitochondrial chaperonin 10, but not groES, is functionally compatible with the heptameric chaperonin 60 of mammalian mitochondria.

ADR1c mutations enhance the ability of ADR1 to activate transcription by a mechanism that is independent of effects on cyclic AMP-dependent protein kinase phosphorylation of Ser-230.

Four ADR1c mutations that occur close to Ser-230 of the Saccharomyces cerevisiae transcriptional activator ADR1 and which greatly enhance the ability of ADR1 to activate ADH2 expression under glucose-repressed conditions have been shown to reduce or eliminate cyclic AMP-dependent protein kinase (cAPK) phosphorylation of Ser-230 in vitro. In addition, unregulated cAPK expression in vivo blocks ADH2 depression in an ADR1-dependent fashion in which ADR1c mutations display decreased sensitivity to unregulated cAPK activity. Taken together, these data have suggested that ADR1c mutations enhance ADR1 activity by blocking cAPK phosphorylation and inactivation of Ser-230. We have isolated and characterized an additional 17 ADR1c mutations, defining 10 different amino acid changes, that were located in the region defined by amino acids 227 through 239 of ADR1. Three observations, however, indicate that the ADR1c phenotype is not simply equivalent to a lack of cAPK phosphorylation. First, only some of these newly isolated ADR1c mutations affected the ability of yeast cAPK to phosphorylate corresponding synthetic peptides modeled on the 222 to 234 region of ADR1 in vitro. Second, we observed that strains lacking cAPK activity did not display enhanced ADH2 expression under glucose growth conditions. Third, when Ser-230 was mutated to a nonphosphorylatable residue, lack of cAPK activity led to a substantial increase in ADH2 expression under glucose-repressed conditions. Thus, while cAPK controls ADH2 expression and ADR1 is required for this control, cAPK acts by a mechanism that is independent of effects on ADR1 Ser-230. It was also observed that deletion of the ADR1c region resulted in an ADR1c phenotype. The ADR1c region is, therefore, involved in maintaining ADR1 in an inactive form. ADR1c mutations may block the binding of a repressor to ADR1 or alter the structure of ADR1 so that transcriptional activation regions become unmasked.

Characterization of the adr1-1 nonsense mutation identifies the translational start of the yeast transcriptional activator ADR1.

We have characterized a nonsense mutation in the ADR1 gene that identifies the translational start of the ADR1 protein. The ADR1 gene of Saccharomyces cerevisiae is required for synthesis of the glucose-repressible alcohol dehydrogenase (ADH2). The adr1-1 mutation, which inhibits ADH2 expression, was identified as a C to G transversion at base pair +32. This alteration would result in a UGA nonsense codon in place of a serine codon that would lead to termination of the ADR1 polypeptide after the 10th amino acid. The effect of the adr1-1 mutation was partially reversed by UGA-tRNA suppressors, indicating that the adr1-1 mutation affects ADR1 expression at the translational level. These observations establish that the first available AUG in the ADR1 sequence is used as the translational start site of ADR1. Tyrosine or leucine UGA-tRNA-suppressors resulted in levels of adr1-1 activity similar to that found for a serine UGA-tRNA-suppressor, suggesting that serine residue-11 is not essential to ADR1 function. Northern analyses showed that the 5.1 kb ADR1 mRNA was two- to three-fold more abundant when isolated from a strain carrying the ADR1 allele than from an isogenic strain containing the adr1-1 allele. These data confirm that the 5.1 kb mRNA is the ADR1 mRNA and suggest that inhibition of adr1-1 mRNA translation results in more rapid degradation of the adr1-1 mRNA.

Identification of functional regions in the yeast transcriptional activator ADR1.

The transcriptional activator ADR1 from Saccharomyces cerevisiae is a postulated DNA-binding protein that controls the expression of the glucose-repressible alcohol dehydrogenase (ADH2). Carboxy-terminal deletions of the ADR1 protein (1,323 amino acids in length) were used to localize its functional regions. The transcriptional activation region was localized to the N-terminal 220 amino acids of ADR1 containing two DNA-binding zinc finger motifs. In addition to the N terminus, a large part of the ADR1 sequence was shown to be essential for complete activation of ADH2. Deletion of the putative phosphorylation region, defined by ADR1c mutations that overcome glucose repression, did not render ADH2 expression insensitive to glucose repression. Instead, this region (amino acids 220 through 253) was found to be required by ADR1 to bypass glucose repression. These results suggest that ADR1c mutations enhance ADR1 function, rather than block an interaction of the putative phosphorylation region with a repressor molecule. Furthermore, the protein kinase CCR1 was shown to affect ADH2 expression when the putative phosphorylation region was removed, indicating that CCR1 does not act solely through this region. A functional ADR1 gene was also found to be necessary for growth on glycerol-containing medium. The N-terminal 506 amino acids of ADR1 were required for this newly identified function, indicating that ADH2 activation and glycerol growth are controlled by separate regions of ADR1.