A urokinase receptor promoter element (-152/-135) bound with an AP-2-alpha-related protein and SP1 is required for the induction of gene expression by PMA and SRC

The urokinase-type plasminogen activator receptor (u-PAR) promotes extracellular matrix degradation, invasion and metastasis. A first objective of this dissertation was to identify cis-elements and trans-acting factors activating u-PAR gene expression through a previously footprinted (–148/–124) promoter region. Mobility shifting experiments on nuclear extracts of a high u-PAR-expressing colon cancer cell line (RKO) indicated Sp1, Sp3 and a factor similar to, but distinct from, AP-2α bound to an oligonucleotide spanning –152/–135. Mutations preventing the binding of the AP-2α-related factor reduced u-PAR promoter activity. In RKO, the expression of a dominant negative AP-2 (AP-2αB) diminished u-PAR promoter activity, protein and u-PAR mediated laminin degradation. Conversely, u-PAR promoter activity in low u-PAR-expressing GEO cells was increased by AP-2αA expression. PMA treatment, which induces u-PAR expression, caused an increased amount of the AP-2α-related factor-containing complex in GEO, and mutations preventing AP-2α-like and Sp1/Sp3 binding reduced the u-PAR promoter stimulation by PMA. In resected colon cancers, u-PAR protein amounts were related to the amount of the AP-2α-related factor-containing complex. In conclusion, constitutive and PMA- inducible u-PAR gene expression and -proteolysis are mediated partly through transactivation via a promoter sequence (–152/435) bound with an AP-2α-related factor and Sp1/Sp3. ^ A second interest of this dissertation was to determine if a constitutively active Src regulates the transcription of the u-PAR gene, since c-src expression increases invasion in colon cancer. Increased u-PAR protein and laminin degradation paralleling elevated Src activity was evident in SW480 colon cancer cells stably expressing a constitutively active Src (Y- c-src527F). Nuclear run-on experiments indicated that this was due largely to transcriptional activation. While transient transfection of SW480 cells with Y-c-src527F induced a u-PAR-CAT-reporter, mutations preventing Sp1-binding to promoter region –152/435 abolished this induction. Mobility shift assays revealed increased Sp1 binding to region –152/135 with nuclear extracts of Src-transfected SW480 cells. Finally, the amounts of endogenous u-PAR in resected colon cancers significantly correlated with Src-activity. These data suggest that u-PAR gene expression and proteolysis are regulated by Src, this requiring the promoter region (–152/–135) bound with Sp1, thus, demonstrating for the first time that transcription factor Sp1 is a downstream effector of Src. ^

Mechanism of IFN-induced apoptosis and sensitization by the proteasome inhibitor bortezomib in bladder cancer cells

Bladder cancer is the fifth most common cancer with more than 50,000 cases diagnosed each year. Interferon-α (IFNα) is mostly used in combination with BCG for the treatment of transitional cell carcinoma (TCC). To examine the effects of IFNα on bladder cancer cells, I analyzed a panel of 20 bladder cancer cell lines in terms of their sensitivity to IFNα-induced apoptosis and the underlying mechanisms. I identified three categories: cells that die after 48hr, after 72h, and cells resistant even after 72hr of IFNα treatment. Examination of the IFN-signal transduction pathway revealed that the defect was not due to abrogation of IFN signaling. Further analysis demonstrated dependency of IFN-induced apoptosis on caspase-8, implicating the role of death receptors in IFN-induced cell death. Of the six most-IFN-sensitive cell lines, the majority upregulated Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) at the mRNA and protein level and IFN-induced cell death was mediated through TRAIL, while a minority of the most IFN-sensitive cells undergo apoptosis through a TNFα-dependent mechanism. IFNα resistance was due to either absence of TRAIL upregulation at the mRNA or protein level, resistance to exogenous rhTRAIL itself or lack of sensitization to IFN-induced cell death. Downregulation of XIAP, or XIAP inactivation through its regulator NFκB has been reported to sensitize tumor cells to death receptor-induced cell death. Baseline and IFN-inducible XIAP levels were examined in the most and least IFN-sensitive cells, knocking down XIAP and the p65 subunit of NFκB enhanced IFN-induced cell death, implicating XIAP downregulation as a mechanism through which bladder cancer cells are sensitized to IFN-induced apoptosis. To determine whether or not the proteasome inhibitor Bortezomib (BZ) sensitizes bladder cancer cells to IFN-induced cell death, the combined effects of IFN+BZ and the underlying molecular mechanisms were examined both in vitro and in vivo using two bladder xenograft models. In both models, tumor growth inhibition was the result of either increased cell death of tumor cells exerted by the two agents and/or inhibition of angiogenesis. In vitro, MAP downregulation in response to the combined treatment of IFN+BZ accounts for one of the mechanisms mediating IFN+BZ cell death in bladder cancer cells. ^

A cellular cleavage pathway for the Moloney murine leukemia virus precursor protein, Pr65$\rm\sp{gag}$: Identification of a new viral protein containing CAp30 and NCp10 sequences

The origin and structure of P55$\sp{\rm gag},$ a gag encoded polyprotein lacking the nucleocapsid protein, NCp10, have been explored. Evidence shows that P55$\sp{\rm gag}$ is formed by non-viral proteolytic cleavage of the Moloney murine leukemia virus (MoMuLV)gag precursor protein, Pr65$\sp{\rm gag}.$ P55$\sp{\rm gag}$ is produced in cells infected by a viral protease deletion mutant and by a recombinant murine sarcoma virus known to lack the protease gene, implying that a cellular protease is responsible for the cleavage. Structural and immunological studies show that the protein cleavage site is upstream of the CAp30-NCp10 viral proteolytic junction, implying that P55$\sp{\rm gag}$ lacks the carboxy-terminal residues of CAp30. During the course of studying P55$\sp{\rm gag},$ another protein was discovered, which I named nucleocapsid-related protein(NCRP). NCRP possesses the portion of CAp30 that is lacking in P55$\sp{\rm gag}.$ NCRP possesses antigenic epitopes present in CAp30 and NCp10. NCRP was observed in virus lysates and in nuclear lysates of MoMuLV infected cells; it was not detected in the cytoplasmic fractions of MoMuLV infected cells. Our results indicated that NCRP originates from Pr65$\sp{\rm gag},$ resulting from the same cellular proteolytic cleavage event that produces the viral cellular protein P55$\sp{\rm gag}.$ P55$\sp{\rm gag}$- and NCRP-like proteins also were observed in AKV murine leukemia virus (AKV MuLV) and feline leukemia virus (FeLV) infected cells and in their respective virus particles. The site of cleavage that yields P55$\sp{\rm gag}$ and NCRP is within the carboxy terminus of CAp30, likely within a motif highly conserved among mammalian type C retroviruses. This new motif, called the capsid conserved motif (CCM), overlaps a region containing both a possible bipartite nuclear targeting sequence and a region homologous with the U1 small nuclear ribonucleoprotein 70-kD protein. This domain, when intact, may act as a nuclear targeting sequence for the gag precursor proteins Pr65$\sp{\rm gag}$ and CAp30. Nuclei of cells infected with MoMuLV were examined for the presence of gag proteins. Both Pr65$\sp{\rm gag}$ and CAp30 were detected in the nuclear fraction of MoMuLV, AKV MuLV and FeLV infected cells. P55$\sp{\rm gag}$ was never detected in the nucleus of MoMuLV, AKV MuLV and FeLV infected cells or in their respective virus particles. I propose that NCRP may be involved in sequestering viral genomic RNA for the purposes of encapsidation and intracellular viral genomic RNA dimerization. ^

MOUSE MAMMARY TUMOR VIRUS-RELATED PROTEINS: ISOLATION AND CHARACTERIZATION OF A UNIQUE GLYCOPROTEIN

Mouse mammary tumor virus (MMTV) contained six major proteins, identified as gp55, gp33, p25, pp20, p12, and p10. Immunoprecipitation of cytoplasmic extracts from MMTV-infected, pulse-labeled cells identified three MMTV core-specific precursor proteins, termed Pr78('gag), Pr110('gag), Pr110('gag), and Pr180('gag+). The major intracellular core-specific precursor polyprotein, Pr78('gag), contained antigenic determinants and tryptic peptides characteristic of p25, p12, and p10. Pr110('gag) contained all but one of the leucine-containing tryptic peptides of Pr78('gag), plus several additional peptides. In addition to Pr78('gag) and Pr110('gag), monospecific antisera to virion p12 and p25 also precipitated from pulse-labeled cells a small amount of Pr180('gag+). This large polyprotein contained nearly all of the leucine-containing tryptic peptides of Pr78('gag) and Pr110('gag) plus several additional peptides. By analogy to type-C viral systems, Pr180('gag+) is presumed to represent a gag-pol-specific common precursor which is the major translation product in the synthesis of MMTV RNA-dependent-DNA polymerase. Immunoprecipitation of cytoplasmic extracts from pulse-labeled cells with antisera to gp55 identified two envelope-specific proteins, designated gPr76('env) and gP79('env). The major envelope-specific precursor, gPr76('env), could be labeled with radioactive glucosamine and contained antigenic determinants and tryptic peptides characteristic of gp55 and gp33. A quantitatively minor glycoprotein, gP79('env), contained both fucose and glucosamine and was precipitable from cytoplasmic extracts with monospecific serum to gp55. It is suggested that gP79('env) represents fucosylated gPr76('env) which is transiently synthesized and cleaved rapidly into gp55 and gp33.^ A glycoprotein of 130,00 molecular weight (gP130) was precipitable from the cytoplasm of GR-strain mouse mammary tumor cells by a rabbit antiserum (anti-MMTV) to Gr-strain mouse mammary tumors virus (GR-MMTV). Two dimensional thin layer analysis of ('35)S-methionine-containing peptides revealed that five of nine gp33 peptides and one of seven gp55 peptides were shared by gP130 and gPr76('env). Six of ten p25 peptides and four more core-related peptides were shared by Pr78('gag) and gP130. Protein gP130 also contained several tryptic peptides not found in gPr76('env), or in the core protein precursors Pr78('gag), Pr110('gag), or Pr180('gag+). both gP130 and a second protein, p30, were found in immunoprecipitates of detergent disrupted, isotopically labeled GR-MMTV treated with anti-MMTV serum. Results suggest that antibodies to gP130 in the anti-MMTV serum are capable of recognizing those protein sequences which are not related to viral structural proteins. These gP130-unique peptides are evidently host specific. Polyproteins consisting of juxtaposed host- and virus-related protein tracts have been implicated in the process of cell transformation in other mammalian systems. Therefore, gP130 may be instrinsic to the oncogenic potential of MMTV. ^

The transcription factor nuclear factor kappa B (NFkappaB) maintains TRAIL resistance in human pancreatic cancer cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/Apo2L) is a member of the TNF family of cytokines that induces apoptosis in a variety of tumor cells while sparing normal cells. However, many human cancer cell lines display resistance to TRAIL-induced apoptosis and the mechanisms contributing to resistance remain controversial. Previous studies have demonstrated that the dimeric transcription factor Nuclear Factor kappa B (NFκB) is constitutively active in a majority of human pancreatic cancer cell lines and primary tumors, and although its role in tumor progression remains unclear it has been suggested that NFκB contributes to TRAIL resistance. Based on this, I examined the effects of NFκB inhibitors on TRAIL sensitivity in a panel of nine pancreatic cancer cell lines. I show here that inhibitors of NFκB, including two inhibitors of the proteasome (bortezomib (Velcade™, PS-341) and NPI-0052), a small molecule inhibitor of IKK (PS1145), and a novel synthetic diterpene NIK inhibitor (NPI-1342) reverse TRAIL resistance in pancreatic cancer cell lines. Further analysis revealed that the expression of the anti-apoptosic proteins BclXL and XIAP was significantly decreased following exposure to these inhibitors alone and in combination with TRAIL. Additionally, treatment with NPI0052 and TRAIL significantly reduced tumor burden relative to the control tumors in an L3.6pl orthotopic pancreatic xenograft model. This was associated with a significant decrease in proliferation and an increase in caspase 3 and 8 cleavage. Combination therapy employing PS1145 or NPI-1342 in combination with TRAIL also resulted in a significant reduction in tumor burden compared to either agent alone in a Panc1 orthotopic xenograft model. My studies show that combination therapy with inhibitors of NFκB alone and TRAIL is effective in pre-clinical models of pancreatic cancer and suggests that the approach should be evaluated in patients. ^

NOVEL THERAPEUTIC TARGETS IDENTIFIED BY HIGH-THROUGHPUT TECHNOLOGIES FOR TRIPLE-NEGATIVE BREAST CANCER

Triple-negative breast cancers (TNBC) are characterized by the lack of or reduced expression of the estrogen and progesterone receptors, and normal expression of the human epidermal growth factor receptor 2. The lack of a well-characterized target for treatment leaves only systemic chemotherapy as the mainstay of treatment. Approximately 60-70% of patients are chemosensitive, while the remaining majority does not respond. Targeted therapies that take advantage of the unique molecular perturbations found in triple-negative breast cancer are needed. The genes that are frequently amplified or overexpressed represent potential therapeutic targets for triple-negative breast cancer. The purpose of this study was to identify and validate novel therapeutic targets for triple-negative breast cancers. 681 genes showed consistent and highly significant overexpression in TNBC compared to receptor-positive cancers in 2 data sets. For two genes, 3 of the 4 siRNAs showed preferential growth inhibition in TNBC cells. These two genes were the low density lipoprotein receptor-related protein 8 (LRP8) and very low-density lipoprotein receptor (VLDLR). Exposure to their cognate ligands, reelin and apolipoprotein E isoform 4 (ApoE4), stimulated the growth of TNBC cells in vitro. Suppression of the expression of either LRP8 or VLDLR or exposure to RAP (an inhibitor of ligand binding to LRP8 and VLDLR) abolished this ligand-induced proliferation. High-throughput protein and metabolic arrays revealed that ApoE4 stimulation rescued TNBC cells from serum-starvation induced up-regulation of genes involved in lipid biosynthesis, increased protein expression of oncogenes involved in the MAPK/ERK and DNA repair pathways, and reduced the serum-starvation induction of biochemicals involved in oxidative stress response and glycolytic metabolism. shLRP8 MDA-MB-231 xenografts had reduced tumor volume, in comparison to parental and shCON xenografts. These results indicate that LRP8-APOE signaling confers survival advantages to TNBC tumors under reduced nutrient conditions and during cellular environmental stress. We revealed that the LRP8-APOE receptor-ligand system is overexpressed in human TNBC. We also demonstrated that this receptor system mediates a strong growth promoting and survival function in TNBC cells in vitro and helps to sustain the growth of MDA-MD-231 xenografts. We propose that inhibitors of LRP8-APOE signaling may be clinically useful therapeutic agents for triple-negative breast cancer.

The transcriptional regulation of the {\it neu\/} gene: Examples of activation, repression, and cell-specific expression

The neu oncogene encodes a growth factor receptor-like protein, p185, with an intrinsic tyrosine kinase activity. A single point mutation, an A to T transversion resulting in an amino acid substitution from valine to glutamic acid, in the transmembrane domain of the rat neu gene was found to be responsible for the transforming and tumorigenic phenotype of the cells that carry it. In contrast, the human proto-neu oncogene is frequently amplified in tumors and cell lines derived from tumors and the human neu gene overexpression/amplification in breast and ovarian cancers is known to correlate with poor patient prognosis. Examples of the human neu gene overexpression in the absence of gene amplification have been observed, which may suggest the significant role of the transcriptional and/or post-transcriptional control of the neu gene in the oncogenic process. However, little is known about the transcriptional mechanisms which regulate the neu gene expression. In this study, three examples are presented to demonstrate the positive and negative control of the neu gene expression.^ First, by using band shift assays and methylation interference analyses, I have identified a specific protein-binding sequence, AAGATAAAACC ($-$466 to $-$456), that binds a specific trans-acting factor termed RVF (for EcoRV factor on the neu promoter). The RVF-binding site is required for maximum transcriptional activity of the rat neu promoter. This same sequence is also found in the corresponding regions of both human and mouse neu promoters. Furthermore, this sequence can enhance the CAT activity driven by a minimum promoter of the thymidine kinase gene in an orientation-independent manner, and thus it behaves as an enhancer. In addition, Southwestern (DNA-protein) blot analysis using the RVF-binding site as a probe points to a 60-kDa polypeptide as a potential candidate for RVF.^ Second, it has been reported that the E3 region of adenovirus 5 induces down-regulation of epidermal growth factor (EGF) receptor through endocytosis. I found that the human neu gene product, p185, (an EGF receptor-related protein) is also down-regulated by adenovirus 5, but via a different mechanism. I demonstrate that the adenovirus E1a gene is responsible for the repression of the human neu gene at the transcriptional level.^ Third, a differential expression of the neu gene has been found in two cell model systems: between the mouse fibroblast Swiss-Webster 3T3 (SW3T3) and its variant NR-6 cells; and between the mouse liver tumor cell line, Hep1-a, and the mouse pancreas tumor cell line, 266-6. Both NR-6 and 266-6 cell lines are not able to express the neu gene product, p185. I demonstrate that, in both cases, the transcriptional repression of the neu gene may account for the lack of the p185 expression in these two cell lines. ^

Functional analysis of the sea urchin {\it orthodenticle\/} -related gene, {\it SPOTX\/}, in aboral ectoderm-specific gene activation and aboral ectoderm cell fate specification

An important question in developmental biology is how embryonic cell types are derived from a fertilized egg. To address this question, this thesis investigates the mechanisms by which the aboral ectoderm-specific Spec2a gene is spatially and temporally regulated during sea urchin embryogenesis. The Spec2a gene of the sea urchin Strongylocentratus purpuratus has served as a valuable maker to understand the basis of lineage-specific gene activation and the role of transcription factors in cell fate specification. The hypothesis is that transcription factors responsible for cell type-specific gene activation are key components in the initial cell specification step. The Spec2a gene, which encodes a small cytosolic calcium-binding protein, is expressed exclusively in aboral ectoderm cell lineages. The 1516-bp control region of the Spec2a gene contains a 188-bp enhancer element required for temporal activation and aboral ectoderm/mesenchyme cell expression, while an unidentified element upstream of the enhancer represses expression in mesenchyme cells. Using an enhancer activation assay, combined with site-directed mutagenesis, I showed that three TAATCC/T sites within the enhancer are responsible for enhancer activity. Mutagenizing these sites and a fourth one just upstream abolished all activity from the Spec2a control region. A 77-bp DNA fragment from the Spec2a enhancer containing two of the TAATCC/T sites is sufficient for aboral ectoderm/mesenchyme cell expression. A cDNA encoding SpOtx, an orthodenticle-related protein, was cloned from S. purpuratus and shown to bind with high affinity to the TAATCC/T sequences within the Spec2a control region. SpOtx transcripts were found initially in all cells of the cleaving embryo, but they gradually became restricted to oral ectoderm and endoderm cells, suggesting that SpOtx might play a role in the initial temporal activation of the Spec2a gene and most likely has additional functions in the developing embryo. To reveal the broader biological functions of SpOtx, I injected SpOtx mRNA into living sea urchin eggs to determine what effects overexpressing the SpOtx protein might have on embryo development. SpOtx mRNA-injected embryos displayed dramatic alterations in development. Instead of developing into pluteus larvae with 15 different cell types, uniform epithelia balls were formed. These balls consisted of a thin layer of squamous cells with short cilia highly reminiscent of aboral ectoderm. Immunohistochemical staining and RT-PCR demonstrated that the SpOtx-injected embryoids expressed aboral ectoderm markers uniformly, but showed very weak or no expression of markers for non-aboral ectoderm cell types. These data strongly suggested that overexpression of SpOtx redirected the normal fate of non-aboral ectoderm cells to that of aboral ectoderm. These results show that SpOtx is involved in aboral ectoderm differentiation by activating aboral ectoderm-specific genes and that modulating its expression can lead to changes in cell fate. ^

Functions of GCN5 and P /CAF acetyltransferases in mammalian development

Histone acetyltransferases are important chromatin modifiers that function as transcriptional co-activators. The identification of the transcriptional regulator GCN5 as the first nuclear histone acetyltransferase in yeast directly linked chromatin remodeling to transcriptional regulation. Although emerging evidence suggests that acetyltransferases participate in multiple cellular processes, their roles in mammalian development remain undefined. In this study, I have cloned and characterized the mouse homolog of GCN5 and a closely related protein P/CAF that interacts with p300/CBP. In contrast to yeast GCN5, but similar to P/CAF, mouse GCN5 possesses an additional N-terminal domain that confers the ability to acetylate nucleosomal histones. GCN5 and P/CAF exhibit identical substrate specificity and both interact with p300/CBP. Interestingly, expression levels of GCN5 and P/CAF display a complementary pattern in mouse embryos and in adult tissues, suggesting that they have distinct tissue or developmental stage specific roles. To define the in vivo function of GCN5 and P/CAF, I have generated mice that are nullizygous for GCN5 or P/CAF. P/CAF null mice are viable and fertile with no gross morphological defects, indicating that P/CAF is dispensable for development and p300/CBP function in vivo. In contrast, mice lacking GCN5 die between 10.5–11 days of gestation. GCN5 null mice are severely retarded but have anterior ectopic outgrowth. Molecular marker analyses reveal that early mesoderm is formed in GCN5 null mice but further differentiation into distinct mesodermal lineages is perturbed. While presomitic mesoderm and chodamesoderm are missing in GCN5 mutant mice, extraembryonic tissues and lateral mesoderm are unaffected. This is consistent with our finding that GCN5 expression is absent in the heart and extraembryonic tissues but is uniform throughout the rest of the embryo. Remarkably, GCN5 mutant mice exhibit an unusually high incidence of apoptosis in the embryonic ectoderm and mesoderm. Finally, mice doubly null for GCN5 and P/CAF die much earlier than mice harboring the GCN5 mutation alone, suggesting that P/CAF and GCN5 share some overlapping function during embryogenesis. This work is the first study to show that specific acetyltransferase is important for cell survival as well as mesoderm differentiation or maintenance during early mammalian development. ^

Characterization of the NOP -1 opsin photoreceptor in the filamentous fungus Neurospora crassa

Light absorption is an important process for energy production and sensory perception in many organisms. In the filamentous fungus, Neurospora crassa, blue-light is an important regulator of both asexual and sexual development, but the identity of the blue-light receptor is unknown. The work presented in this dissertation initiated the characterization of the putative N. crassa opsin photoreceptor, NOP-1. Opsins were thought to exist only in the archaea and mammals until the discovery of nop-1. All opsins have the same conserved structure of seven transmembrane helical domains with a lysine residue in the seventh helix specific for forming a Schiff-base linkage with retinal. The predicted NOP-1 protein sequence is equally similar to archaeal rhodopsins and a newly identified fungal opsin-related protein group (ORPs). ORPs maintain the seven transmembrane helical structure of opsins, but lack the conserved lysine residue for binding retinal. An ORP gene, orp-1 was identified in N. crassa and this work includes the cloning and sequence analysis of this gene. Characterization of NOP-1 function in N. crassa development began with the construction of a Δnop-1 deletion mutant. Extensive phenotypic analysis of Δnop-1 mutants revealed only subtle defects during development primarily under environmental conditions that induce a stress response. NOP-1 was overexpressed in the heterologous system Pichia pastoris, and it was demonstrated that NOP-1 protein bound all-trans retinal to form a green-light absorbing pigment (λmax = 534 nm) with a photochemical reaction cycle similar to archaeal sensory rhodopsins. nop-1 gene expression was monitored during N. crassa development. nop-1 transcript is highly expressed during asexual sporulation (conidiation) and transcript levels are abundant in the later stages of conidial development. nop-1 expression is not regulated by blue-light or elevated temperatures. Potential functions for NOP-1 were discovered through the transcriptional analysis of conidiation-associated genes in Δnop-1 mutants. NOP-1 exhibits antagonistic transcriptional regulation of conidiation-associated genes late in conidial development, by enhancing the carotenogenic gene, al-2 and repressing the conidiation-specific genes, con-10 and con-13. ^

Enhanced estrogen-induced proliferation in obese rat endometrium.

OBJECTIVE: We tested the hypothesis that the proliferative estrogen effect on the endometrium is enhanced in obese vs lean animals. STUDY DESIGN: Using Zucker fa/fa obese rats and lean control, we examined endometrial cell proliferation and the expression patterns of certain estrogen-regulated proproliferative and antiproliferative genes after short-term treatment with estradiol. RESULTS: No significant morphologic/histologic difference was seen between the obese rats and the lean rats. Estrogen-induced proproliferative genes cyclin A and c-Myc messenger RNA expression were significantly higher in the endometrium of obese rats compared with those of the lean control. Expression of the antiproliferative gene p27Kip1 was suppressed by estrogen treatment in both obese and lean rats; however, the decrease was more pronounced in obese rats. Estrogen more strongly induced the antiproliferative genes retinaldehyde dehydrogenases 2 and secreted frizzled-related protein 4 in lean rats but had little or no effect in obese rats. CONCLUSION: Enhancement of estrogen-induced endometrial proproliferative gene expression and suppression of antiproliferative gene expression was seen in the endometrium of obese vs lean animals.

PAT proteins, an ancient family of lipid droplet proteins that regulate cellular lipid stores.

The PAT family of lipid droplet proteins includes 5 members in mammals: perilipin, adipose differentiation-related protein (ADRP), tail-interacting protein of 47 kDa (TIP47), S3-12, and OXPAT. Members of this family are also present in evolutionarily distant organisms, including insects, slime molds and fungi. All PAT proteins share sequence similarity and the ability to bind intracellular lipid droplets, either constitutively or in response to metabolic stimuli, such as increased lipid flux into or out of lipid droplets. Positioned at the lipid droplet surface, PAT proteins manage access of other proteins (lipases) to the lipid esters within the lipid droplet core and can interact with cellular machinery important for lipid droplet biogenesis. Genetic variations in the gene for the best-characterized of the mammalian PAT proteins, perilipin, have been associated with metabolic phenotypes, including type 2 diabetes mellitus and obesity. In this review, we discuss how the PAT proteins regulate cellular lipid metabolism both in mammals and in model organisms.

Contribution of activity and skin temperature to the risk of developing pressure ulcers in nursing facility residents

Purpose. The central concepts in pressure ulcer risk are exposure to external pressure caused by inactivity and tissue tolerance to pressure, a factor closely related to blood flow. Inactivity measures are effective in predicting pressure ulcer risk. The purpose of the study is to evaluate whether a physiological measure of skin blood flow improves pressure ulcer risk prediction. Skin temperature regularity and self-similarity, as proxy measures of blood flow, and not previously described, may be undefined pressure ulcer risk factors. The specific aims were to determine whether a sample of nursing facility residents at high risk of pressure ulcers classified using the Braden Scale for Pressure Sore Risk© differ from a sample of low risk residents according to (1) exposure to external pressure as measured by resident activity, (2) tissue tolerance to external pressure as measured by skin temperature, and (3) skin temperature fluctuations and recovery in response to a commonly occurring stressor, bathing and additionally whether (4) scores on the Braden Scale mobility subscale score are related to entropy and the spectral exponent. ^ Methods. A two group observational time series design was used to describe activity and skin temperature regularity and self-similarity, calculating entropy and the spectral exponent using detrended fluctuation analysis respectively. Twenty nursing facility residents wore activity and skin temperature monitors for one week. One bathing episode was observed as a commonly occurring stressor for skin temperature.^ Results. Skin temperature multiscale entropy (MSE), F(1, 17) = 5.55, p = .031, the skin temperature spectral exponent, F(1, 17) = 6.19, p = .023, and the activity mean MSE, F(1, 18) = 4.52, p = .048 differentiated the risk groups. The change in skin temperature entropy during bathing was significant, t(16) = 2.55, p = .021, (95% CI, .04-.40). Multiscale entropy for skin temperature was lowest in those who developed pressure ulcers, F(1, 18) = 35.14, p < .001.^ Conclusions. This study supports the tissue tolerance component of the Braden and Bergstrom conceptual framework and shows differences in skin temperature multiscale entropy between pressure ulcer risk categories, pressure ulcer outcome, and during a commonly occurring stressor. ^

Mechanisms of multidrug resistance: Differences between natural and acquired adriamycin -resistant human colon carcinoma cells

Mechanisms of multidrug resistance (MDR) were studied in two independent MDR sublines (AdR1.2 and SRA1.2) derived from the established human colon carcinoma cell line LoVo. AdR1.2 was developed by long-term continuous exposure of the cells to adriamycin (AdR) in stepwise increments of concentration, while SRA1.2 was selected by repetitive pulse treatments with AdR at a single concentration. In this dissertation, the hypothesis that the mechanism of drug resistance in SRA1.2 is different than that in AdR1.2 is tested. While SRA1.2 demonstrated similar biological characteristics when compared to the parental LoVo, AdR1.2 exhibited remarkable alterations in biological properties. The resistance phenotype of AdR1.2 was reversible when the cells were grown in the drug-free medium whereas SRA1.2 maintained its resistance for at least 10 months under similar conditions. Km and Vmax of carrier-mediated facilitated diffusion AdR transport were similar among the three lines. However, both resistant sublines exhibited an energy-dependent drug efflux. AdR1.2 appeared to possess an activated efflux pump, and a decreased nucleus-binding of AdR, whereas SRA1.2 showed merely a lower affinity in binding of AdR to the nuclei. Southern blot analysis showed no amplification of the MDR1 gene in either of the two resistant subclones. However, Western blot analysis using the C219 monoclonal antibody against P170 glycoprotein detected a Mr 150-kDa plasma protein (P150) in AdR1.2 but not in SRA1.2 or in the parental LoVo. In vitro phosphorylation studies revealed that P150 was a phosphoprotein; its phosphorylation was Mg$\sp{2+}$-dependent and could be enhanced by verapamil, an agent capable of increasing intracellular AdR accumulation in AdR1.2 cells. The phosphorylation studies also revealed elevated phosphorylation of a Mr 66-kDa plasma membrane protein that was detectable in the AdR1.2 revertant and in AdR1.2 when verapamil was present, suggesting that hyperphosphorylation of the Mr 66-kDa protein may be related to the reversal of MDR. SDS-PAGE of the plasma membrane protein demonstrated overproduction of a Mr 130-kDa, MDR-related protein in both the resistant sublines. The Mr 130-kDa, MDR-related protein in both the resistant sublines. The Mr 130-kDa protein was not immunoreactive with C219, but its absence in the AdR1.2 revertant and the parental LoVo suggests that it is an MDR-related plasma membrane protein. In conclusion, the results from this study support the author's hypothesis that the mechanisms responsible for "Acquired" and "Natural" MDR are not identical. ^

Multifactor effects and evidence of potential interaction between complement factor H Y402H and LOC387715 A69S in age-related macular degeneration.

BACKGROUND: Variants in the complement cascade genes and the LOC387715/HTRA1, have been widely reported to associate with age-related macular degeneration (AMD), the most common cause of visual impairment in industrialized countries. METHODS/PRINCIPAL FINDINGS: We investigated the association between the LOC387715 A69S and complement component C3 R102G risk alleles in the Finnish case-control material and found a significant association with both variants (OR 2.98, p = 3.75 x 10(-9); non-AMD controls and OR 2.79, p = 2.78 x 10(-19), blood donor controls and OR 1.83, p = 0.008; non-AMD controls and OR 1.39, p = 0.039; blood donor controls), respectively. Previously, we have shown a strong association between complement factor H (CFH) Y402H and AMD in the Finnish population. A carrier of at least one risk allele in each of the three susceptibility loci (LOC387715, C3, CFH) had an 18-fold risk of AMD when compared to a non-carrier homozygote in all three loci. A tentative gene-gene interaction between the two major AMD-associated loci, LOC387715 and CFH, was found in this study using a multiplicative (logistic regression) model, a synergy index (departure-from-additivity model) and the mutual information method (MI), suggesting that a common causative pathway may exist for these genes. Smoking (ever vs. never) exerted an extra risk for AMD, but somewhat surprisingly, only in connection with other factors such as sex and the C3 genotype. Population attributable risks (PAR) for the CFH, LOC387715 and C3 variants were 58.2%, 51.4% and 5.8%, respectively, the summary PAR for the three variants being 65.4%. CONCLUSIONS/SIGNIFICANCE: Evidence for gene-gene interaction between two major AMD associated loci CFH and LOC387715 was obtained using three methods, logistic regression, a synergy index and the mutual information (MI) index.