SFRP4 regulation of Wnt7a signaling and cellular proliferation in the endometrium

In the endometrium, hormonal effects on epithelial cells are often elicited through stromal hormone receptors via unknown paracrine mechanisms. Several lines of evidence support the hypothesis that Wnts participate in stromal-epithelial cell communication and thus mediate hormone action. Characterization of specific Wnt signaling components in the endometrium was performed using cellular localization studies and evaluating hormone effects in a rat model. Wnt7a was expressed in the luminal epithelium, whereas the extracellular Wnt modulator, SFRP4, was localized to the endometrial stroma. SFRP4 expression is significantly decreased in endometrial carcinoma and aberrant Wnt7a signaling has been shown to cause uterine defects and contribute to the onset of disease. The specific Fzds and SFRPs that bind Wnt7a and the particular signal transduction pathway each Wnt7a-Fzd pair activates have not been identified. Additionally, the function of Wnt7a and SFRP4 in the endometrium has not been addressed. A survey of all Wnt signaling proteins expressed in the endometrium was conducted and Fzd5 and Fzd10 were identified as two receptors capable of transducing the Wnt7a signal. Biologically active recombinant Wnt7a and SFRP4 proteins were purified for quantitative biochemical studies. In Ishikawa cells, Wnt7a binding to Fzd5 activated β-catenin/canonical Wnt signaling and increased cellular proliferation. Wnt7a signaling mediated by Fzd10 induced a non-canonical/JNK-responsive pathway. SFRP4 suppressed Wnt7a action in both an autocrine and paracrine manner. Treatment with SFRP4 protein and overexpression of SFRP4 inhibited endometrial cancer cell growth and induced apoptosis in vitro. A split-eGFP complementation assay was developed to visually detect Wnt7a-Fzd interactions and subsequent pathway activation in cells. By employing a unique ELISA-based protein-protein binding technique, it was demonstrated that Wnt7a binds to SFRP4 and Fzd5 with equal nanomolar affinity. The development of these novel biological tools could lead to a better understanding of Wnt-protein interactions and the identification of new modulators of Wnt signaling. This study supports a mechanism by which the nature of the Wnt7a signal in the endometrium is dependent upon the Fzd repertoire of the cell and can be regulated by SFRP4. The potential tumor suppressor function of SFRP4 suggests it may serve as a therapeutic target for endometrial carcinoma. ^

Role of telomere dysfunction, DNA damage response and p53 mutations in tumorigenesis and aging

The ends of eukaryotic chromosomes are protected by specialized ribonucleoprotein structures termed telomeres. Telomeres protect chromosomes from end-to-end fusions, inappropriate repair and degradation. Disruption of this complex activates an ATM/ATR DNA damage response (DDR) pathway. One component of the complex is the Protection Of Telomeres 1 (POT1) protein, an evolutionarily conserved protein which binds single-stranded 3' overhang and is required for both chromosomal end protection and telomere length regulation. The mouse contains two POT1 orthologs, Pot1a and Pot1b. Here we show that both proteins colocalize with telomeres through interaction with the adapter protein TPP1. In addition, compared to Pot1a, the OB-folds of Pot1b possess less sequence specificity for telomeres. Disruption of POT1 proteins result in telomere dysfunction and activation of an ATR-dependent DDR at telomeres, suggesting that this response is normally suppressed by POT1 binding to the single-stranded G-overhang. ^ Telomeres are maintained by telomerase, and its absence in somatic cells results in telomere progressive loss that triggers the activation of p53. Telomere dysfunction initiates genomic instability and induces both p53-dependent replicative senescence and apoptosis to suppress tumorigenesis. In the absence of functional p53, this genomic instability promotes cancer. It was previously not known which aspect of the p53 dependent DNA damage response is important to suppress tumorigenesis initiated by dysfunctional telomeres. The p53R172P knock-in mouse, which is unable to induce apoptosis but retains intact cell cycle arrest/cellular senescence pathways, allowed us to examine whether p53-dependent apoptosis is a major tumor suppression pathway initiated in the setting of telomere dysfunction. Spontaneous tumorigenesis remains potently suppressed in late generation telomerase null mice possessing the p53P/P mutation. These results suggest that suppression of spontaneous tumorigenesis initiated by dysfunctional telomeres requires activation of a p53-dependent senescence pathway. In addition, we used another knock-in mouse model with a p53R172H (p53H) point mutation to test the hypothesis that telomere dysfunction promotes chromosomal instability and accelerates the onset of tumorigenesis in vivo in the setting of this most common gain-of-function mutation in the human Li Fraumeni cancer syndrome. We unexpectedly observed that telomerase null mice possessing dysfunctional telomeres in the setting of the p53H/+ mutation develop significantly fewer tumors, die prematurely and exhibit higher level of cellular senescence, apoptosis and elevated genomic instability compared to telomerase intact p53H/+ and telomerase null p53+/+ mice. These contrasting results thus link cancer and aging to the functional status of telomeres and the integrity of the p53 pathway. ^

The cellular and molecular responses to a novel nucleotide analogue, GS-9219

GS-9219 is a cell-permeable double-prodrug of the acyclic nucleotide analogue 9-(2-phosphonylmethoxyethyl)guanine (PMEG). The conversion of GS-9219 to its active metabolite, PMEG diphosphate (PMEGpp), involves several intracellular enzymatic reactions which reduces the concentration of nephrotoxic PMEG in plasma. PMEGpp competes with the natural substrate, dGTP, for incorporation by DNA polymerases. The lack of a 3'-hydroxyl moiety makes PMEGpp a de facto DNA chain-terminator. The incorporation of PMEGpp into DNA during DNA replication causes DNA chain-termination and stalled replication forks. Thus, the primary mechanism of action of GS-9219 in replicating cells is via DNA synthesis inhibition. GS-9219 has substantial antiproliferative activity against activated lymphocytes and tumor cell lines of hematological malignancies. Tumor cell proliferation was significantly reduced as measured by PET/CT scans in dogs with advanced-stage, spontaneously occurring non-Hodgkin's lymphoma (NHL).^ The hypothesis of this dissertation is that the incorporation of PMEGpp into DNA during repair re-synthesis would result in the inhibition of DNA repair and accumulation of DNA damage in chronic lymphocytic leukemia (CLL) cells and activate signaling pathways to cell death.^ To test this hypothesis, CLL cells were treated with DNA-damaging agents to stimulate nucleotide excision repair (NER) pathways, enabling the incorporation of PMEGpp into DNA. When NER was activated by UV, PMEGpp was incorporated into DNA in CLL cells. Following PMEGpp incorporation, DNA repair was inhibited and led to the accumulation of DNA strand breaks. The combination of GS-9219 and DNA-damaging agents resulted in more cell death than the sum of the single agents alone. The presence of DNA strand breaks activated the phosphatidylinositol 3-kinase-like protein kinase (PIKK) family members ataxia-telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK). The activated ATM initiated signaling to the downstream target, p53, which was subsequently phosphorylated and accumulated to exert its apoptotic functions. P53-targeted pro-apoptotic genes, Puma and Bax, were upregulated and activated when DNA repair was inhibited, likely contributing to cell death. ^

The eukaryotic cellular stress response: Biochemical and genetic analyses in Saccharomyces cerevisiae

All cells must have the ability to deal with a variety of environmental stresses. Failure to correctly adapt to and/or protect against adverse stress conditions can lead to cell death. In humans, stress response defects have been linked to a number of neurodegenerative diseases and cancer, underscoring the importance of developing a fundamental understanding of the eukaryotic stress response.^ In an effort to characterize cellular response to high temperature stress, I identified and described one member of a novel gene family— RTR1. I show that the RTR1 gene and its protein product genetically and biochemically interact with core subunits of the RNA polymerase II enzyme. Appropriately, loss of RTR1 results in defective transcription from multiple promoters. These data provide evidence that Rtr1, which is essential under stress conditions, acts as a key regulator of transcription.^ In addition to transcriptional regulation, cells deal with many stressors by inducing molecular chaperones. Molecular chaperones are ubiquitous in all living cells and bind unfolded or damaged proteins and catalyze refolding or degradation. Hsp90 is a unique chaperone because it targets specific clients—typically signaling proteins—for maturation. While it has been shown that Sse1, the yeast Hsp110, is a critical regulator of the Hsp90 chaperone cycle, this work describes the molecular basis for that regulation. I show that Sse1 modulates Hsp90 function through regulation of Hsp70 nucleotide exchange. Further, Hsp110-type nucleotide exchange factors (NEFs) appear to have a specific role in modulating Hsp90 function in this manner. Finally, in addition to Hsp110, the eukaryotic cytosol contains two other types of Hsp70 NEF: Snl1 (BAG-domain protein) and Fes1 (HspBP1-like protein). I investigated the cellular roles of these NEFs to better understand the reason that eukaryotic cells contain three distinct protein families that perform the same biochemical function. I show that while cytsolic Hsp70 NEFs have some degree of functional overlap, they also exhibit striking divergence. Taken together, the work presented in this dissertation provides a more detailed understanding of the eukaryotic stress response. ^

Role of TRP Channels in Mediating the Calcium Signaling Response of Brain Endothelial Cells to Mechanical Stretch

Traumatic brain injury (TBI) often results in disruption of the blood brain barrier (BBB), which is an integral component to maintaining the central nervous system homeostasis. Recently cytosolic calcium levels ([Ca2+]i), observed to elevate following TBI, have been shown to influence endothelial barrier integrity. However, the mechanism by which TBI-induced calcium signaling alters the endothelial barrier remains unknown. In the present study, an in vitro BBB model was utilized to address this issue. Exposure of cells to biaxial mechanical stretch, in the range expected for TBI, resulted in a rapid cytosolic calcium increase. Modulation of intracellular and extracellular Ca2+ reservoirs indicated that Ca2+ influx is the major contributor for the [Ca2+]i elevation. Application of pharmacological inhibitors was used to identify the calcium-permeable channels involved in the stretch-induced Ca2+ influx. Antagonist of transient receptor potential (TRP) channel subfamilies, TRPC and TRPP, demonstrated a reduction of the stretch-induced Ca2+ influx. RNA silencing directed at individual TRP channel subtypes revealed that TRPC1 and TRPP2 largely mediate the stretch-induced Ca2+ response. In addition, we found that nitric oxide (NO) levels increased as a result of mechanical stretch, and that inhibition of TRPC1 and TRPP2 abolished the elevated NO synthesis. Further, as myosin light chain (MLC) phosphorylation and actin cytoskeleton rearrangement are correlated with endothelial barrier disruption, we investigated the effect mechanical stretch had on the myosin-actin cytoskeleton. We found that phosphorylated MLC was increased significantly by 10 minutes post-stretch, and that inhibition of TRP channel activity or NO synthesis both abolished this effect. In addition, actin stress fibers formation significantly increased 2 minutes post-stretch, and was abolished by treatment with TRP channel inhibitors. These results suggest that, in brain endothelial cells, TRPC1 and TRPP2 are activated by TBI-mechanical stress and initiate actin-myosin contraction, which may lead to disruption of the BBB.

Cellular Uptake of Neutrohpil Elastase Links Inflammation to Adaptive Immunity

Many tumors arise from sites of inflammation providing evidence that innate immunity is a critical component in the development and progression of cancer. Neutrophils are primary mediators of the innate immune response. Upon activation, an important function of neutrophils is release of an assortment of proteins from their granules including the serine protease neutrophil elastase (NE). The effect of NE on cancer has been attributed primarily to its ability to degrade the extracellular matrix thereby promoting invasion and metastasis. Recently, it was shown that NE could be taken up by lung cancer cells leading to degradation of insulin receptor substrate-1 thereby promoting hyperactivity of the phosphatidylinositol-3 kinase (PI3K) pathway and tumor cell proliferation. To our knowledge, nobody has investigated uptake of NE by other tumor types. In addition, NE has broad substrate specificity suggesting that uptake of NE by tumor cells could impact processes regulating tumorigenensis other than activation of the PI3K pathway. Neutrophil elastase has been identified in breast cancer specimens where high levels of NE have prognostic significance. These studies have assessed NE levels in whole tumor lysates. Because the major source of NE is from activated neutrophils, we hypothesized that breast cancer cells do not have endogenous NE but may take up NE released by tumor associated neutrophils in the tumor microenvironment and that this could provide a link between the innate immune response to tumors and specific adaptive immune responses. In this thesis, we show that breast cancer cells lack endogenous NE expression and that they are able to take up NE resulting in increased generation of low molecular weight cyclin E (CCNE) and enhanced susceptibility to lysis by CCNE-specific cytotoxic T lymphocytes. We also show that after taking up NE and proteinase 3 (PR3), a second primary granule protease with significant homology to NE, breast cancer cells cross-present the NE- and PR3-derived peptide PR1 rendering them susceptible to PR1-targeted therapies. Taken together, our data support a role for NE uptake in modulating adaptive immune responses against breast cancer.

Cellular Trafficking of Single and Multistage Vectors

Nanomedicine is an innovative field of science which has recently generated many drug delivery platforms with exciting results. The great potential of these strategies rely on the unique characteristics of the devices at the nano-scale in terms of long time circulation in the blood stream, selective accumulation at the lesions sites, increased solubility in aqueous solutions, etc. Herein we report on a new drug delivery system known as a multistage system which is comprised of non-spherical, mesoporous silicon particles loaded with second stage nanoparticles. The rationally designed particle shape, the possibility to modulate the surface properties and the degree of porosity allow these carriers to be optimized for vascular targeting and to overcome the numerous biological barriers found in drug delivery. In this study we investigated the intra and inter cellular trafficking of the multistage system in endothelial cells bringing evidence of its bio-compatibility as well as its ability to perform multiple intra and inter cellular tasks. Once internalized in cells, the multi-particle construct is able to dissociate, localizing in different subcellular compartments which can be targeted for exocytosis. In particular the second stage nanoparticles were found to be secreted in microvesicles which can act as mediators of transfer of particles across the endothelium and between different endothelial and cancer cells.

Classification of cellular malignancy associated changes in cervical cancer

Cervical cancer is the leading cause of death and disease from malignant neoplasms among women in developing countries. Even though the Pap smear has significantly decreased the number of deaths from cervical cancer in the past years, it has its limitations. Researchers have developed an automated screening machine which can potentially detect abnormal cases that are overlooked by conventional screening. The goal of quantitative cytology is to classify the patient's tissue sample based on quantitative measurements of the individual cells. It is also much cheaper and potentially can take less time. One of the major challenges of collecting cells with a cytobrush is the possibility of not sampling any existing dysplastic cells on the cervix. Being able to correctly classify patients who have disease without the presence of dysplastic cells could improve the accuracy of quantitative cytology algorithms. Subtle morphologic changes in normal-appearing tissues adjacent to or distant from malignant tumors have been shown to exist, but a comparison of various statistical methods, including many recent advances in the statistical learning field, has not previously been done. The objective of this thesis is to use different classification methods applied to quantitative cytology data for the detection of malignancy associated changes (MACs). In this thesis, Elastic Net is the best algorithm. When we applied the Elastic Net algorithm to the test set, we combined the training set and validation set as "training" set and used 5-fold cross validation to choose the parameter for Elastic Net. It has a sensitivity of 47% at 80% specificity, an AUC 0.52, and a partial AUC 0.10 (95% CI 0.09-0.11).^

Dissection of antitumor activity by lymphokine -activated killer cells: Role of FcR+ precursors and obligatory role of tumor necrosis factor-$\alpha$ in antibody -dependent cellular cytotoxicity

Human peripheral blood lymphocytes (PBL) cultured for varying lengths of time in IL-2 are able to mediate antibody independent cellular cytotoxicity (AICC) as well as antibody dependent cellular cytotoxicity (ADCC) against a wide range of tumor targets. The objective of our study is to determine the cytotoxic potential of the subset of LAK cells involved in ADCC, the tumor recognition mechanism in ADCC, the kinetics of ADCC mediated by PBL cultured under various conditions and the role of TNF-$\alpha$ in the development and maturation of ADCC effectors in the LAK population.^ The model system in this study for ADCC used a monoclonal antibody 14G2a (IgG2a), that recognizes the GD2 epitope on human melanoma cell line, SK-Mel-1. The target recognition mechanism operative in AICC (traditionally known as lymphokine activated killing or LAK) is an acquired property of these IL-2 activated cells which confers on them the unique ability to distinguish between tumor and normal cells. This recognition probably involves the presence of a trypsin sensitive N-linked glycoprotein epitope on tumor cells. Proteolytic treatment of the tumor cells with trypsin renders them resistant to AICC by PBL cultured in IL-2. However, ADCC is unaffected. This ADCC, mediated by the relatively small population of cells that are positive for the Fc receptor for IgG (FcR), is an indication that this subset of "LAK" cells does not require the trypsin sensitive epitope on tumor cells to mediate killing. Enriching PBL for FcR+ cells markedly enhanced both AICC and ADCC and also reduced the IL-2 requirement of these cells.^ The stoichiometry of Fc receptor (FcR) expression on the cytotoxic effectors does not correlate with ADCC lytic activity. Although FcRs are necessary to mediate ADCC, other factors, appear to regulate the magnitude of cytolytic activity. In order to investigate these putative factors, the kinetics of ADCC development was studied under various conditions (in IL-2 (10u/ml) and 100u/ml), in IL-2(10u/ml) + TNF$\alpha$ (500u/ml) and in TNF-$\alpha$ (500u/ml) alone). Addition of exogenous TNF-$\alpha$ into the four hour cytotoxicity assay did not increase ADCC, nor did anti-TNF antibodies result in inhibition. On the other hand, addition of anti-TNF antibodies to PBL and IL-2 for 24 hours, resulted in a marked inhibition of the ADCC, suggesting that endogenous TNF-$\alpha$ is obligatory for the maturation and differentiation of ADCC effectors. ^

Design, synthesis and cellular and molecular pharmacology of 2$\sp\prime$-bromo-4$\sp\prime$-epidaunorubicin (WP401): A novel non-cross-resistant anthracycline antibiotic with the intrinsic ability to circumvent P -glycoprotein-mediated drug resistance

We designed and synthesized a novel daunorubicin (DNR) analogue that effectively circumvents P-glycoprotein (P-gp)-mediated drug resistance. The fully protected carbohydrate intermediate 1,2-dibromoacosamine was prepared from acosamine and effectively coupled to daunomycinone in high yield. Deprotection under alkaline conditions yielded 2$\sp\prime$-bromo-4$\sp\prime$-epidaunorubicin (WP401). The in vitro cytotoxicity and cellular and molecular pharmacology of WP401 were compared with those of DNR in a panel of wild-type cell lines (KB-3-1, P388S, and HL60S) and their multidrug-resistant (MDR) counterparts (KB-V1, P388/DOX, and HL60/DOX). Fluorescent spectrophotometry, flow cytometry, and confocal laser scanning microscopy were used to measure intracellular accumulation, retention, and subcellular distribution of these agents. All MDR cell lines exhibited reduced DNR uptake that was restored, upon incubation with either verapamil (VER) or cyclosporin A (CSA), to the level found in sensitive cell lines. In contrast, the uptake of WP401 was essentially the same in the absence or presence of VER or CSA in all tested cell lines. The in vitro cytotoxicity of WP401 was similar to that of DNR in the sensitive cell lines but significantly higher in resistant cell lines (resistance index (RI) of 2-6 for WP401 vs 75-85 for DNR). To ascertain whether drug-mediated cytotoxicity and retention were accompanied by DNA strand breaks, DNA single- and double-strand breaks were assessed by alkaline elution. High levels of such breaks were obtained using 0.1-2 $\mu$g/mL of WP401 in both sensitive and resistant cells. In contrast, DNR caused strand breaks only in sensitive cells and not much in resistant cells. We also compared drug-induced DNA fragmentation similar to that induced by DNR. However, in P-gp-positive cells, WP401 induced 2- to 5-fold more DNA fragmentation than DNR. This increased DNA strand breakage by WP401 was correlated with its increased uptake and cytotoxicity in these cell lines. Overall these results indicate that WP401 is more cytotoxic than DNR in MDR cells and that this phenomenon might be related to the reduced basicity of the amino group and increased lipophilicity of WP401. ^

Creation of a retinoblastoma mutant with dominant -negative activity

The Retinoblastoma tumor suppressor gene (RB) plays a role in a variety of human cancers. Experimental analyses have indicated that the protein product of the RB gene (pRb) plays a role in cell cycle regulation, and that this protein is required in cellular differentiation, senescence, and cell survival. pRb function is dependent on its ability to bind to cellular factors. There are multiple protein binding domains within pRb. Mutations within these domains which eliminate the ability of pRb to bind its targets result in loss of function. Loss of pRb function leads to tumorigenesis, although uncontrolled cellular proliferation is not a universal response to pRb inactivation. The ultimate response to the loss of pRb is influenced by both the genetic and epigenetic environments. Targeted disruption of RB in mice results in embryonic lethality, demonstrating the requirement for functional pRb in development. Close examination of various tissues from the embryos which lack wildtype RB shows problems in differentiation as well as showing induction of apoptosis. Although disruption of RB has provided useful information, complete inactivation of a gene precludes the possibility of discovering the functions that separate domains may have within the system. Creation of a dominant negative mutant by domain deletion whose phenotype is expressed in the presence of the wildtype may provide information about the intermediate functions of the protein. In addition, tissue specific targeting of a dominant negative mutant of pRb allows for comprehensive analysis of pRb function in organogenesis. In this thesis, a series of RB deletion mutants were created and tested for dominant negative activity as well as cellular localization. A tissue culture assay for dominant negative activity was developed which screens for the phenotype of apoptosis due to loss of pRb function. Two mutants from this series scored positive for dominant negative activity in this assay. The effect of these mutants within the assay environment can be explained by a model in which pRb acts as a facilitator of cell fate pathway decisions. ^

Identification and characterization of cellular genes differentially expressed in a subset of tumors with non -functional WT1

Wilms tumor (WT) or nephroblastoma is a genetically heterogeneous pediatric renal tumor that accounts for 6–7% of all childhood cancers in the U.S. WT1, located at 11p13, is the sole WT gene cloned to date. Additional genomic regions containing genes that play a role in the development of Wilms tumor include 11p15, 7p, 16q, 1p, 17q and 19q. This heterogeneity has made it extremely difficult to develop an understanding of the pathways involved in the development of WT, even in the 5–20% of tumors that show mutations at the WT1 locus. My research addresses this gap in our current comprehension of the development of WT. ^ I have used two complementary approaches to extend the current understanding of molecular changes involved in the development of WT. In order to minimize complexities due to genetic heterogeneity, I confined my analysis to the WT1 pathway by assessing those genetically defined tumors that carry WT1 mutations. WT1 encodes a zinc finger transcription factor, and in vitro studies have identified many genes that are potentially regulated in vivo by WT1. However, there is very little in vivo data that suggests that they are transcriptionally regulated endogenously by WT1. In one approach I assessed the role of WT1 in the in vivo regulation of PDGFA and IGF2, two genes that are strong contenders for endogenous regulation by WT1. Using primary tissue samples, I found no correlation between the level of RNA expression of WT1 with either PDGFA or IGF2, suggesting that WT1 does not play a critical role in their expression in either normal kidney or WT. ^ In a parallel strategy, using differential display analysis I compared global gene expression in a subset of tumors with known homozygous inactivating WT1 mutations (WT1-tumors) to the gene expression in a panel of appropriate control tissues (fetal kidney, normal kidney, rhabdoid tumor and pediatric renal cell carcinoma). Transcripts that are aberrantly expressed in this subset of Wilms tumors are candidates for endogenous transcriptional regulation by WT1 as well as for potentially functioning in the development of WT. By this approach I identified several differentially expressed transcripts. I further characterized two of these transcripts, identifying a candidate WT gene in the process. I then performed a detailed analysis of this WT candidate gene, which maps to 7p. Future studies will shed more light on the role of these differentially expressed genes in WT. ^

Circumvention of cellular defense responses to DNA -directed nucleoside analogues by the protein kinase inhibitor, UCN-01

DNA-directed nucleoside analogues, such as ara-C, fludarabine, and gemcitabine, are antimetabolites effective in the treatment of a variety of cancers. However, resistance to nucleoside analogue-based chemotherapy in treatments is still a major problem in therapy. Therefore, it is essential to develop rationales for optimizing the use of nucleoside analogues in combination with other anticancer drugs or modalities such as radiation. The present study focuses on establishing mechanism-based combination strategy to overcome resistance to nucleoside analogues. ^ I hypothesized that the cytostatic concentrations of nucleoside analogues may cause S-phase arrest by activating an S-phase checkpoint that consists of a series of kinases. This may allow cells to repair damaged DNA over time and spare cytotoxicity. Thus, the ability of cells to enact an S-phase arrest in response to incorporation of potentially lethal amounts of nucleoside analogue may serve as a mechanism of resistance to S-phase-specific agents. As a corollary, the addition of a kinase inhibitor, such as UCN-01, may dysregulate the checkpoint response and abrogate the survival of S-phase-arrested cells by suppression of the survival signaling pathways. Using gemcitabine as a model of S-phase-specific nucleoside analogues in human acute myelogenous leukemia ML-1 cells, I demonstrated that cells arrested in S-phase in response to cytostatic conditions. Proliferation continued after washing the cells into drug-free medium, suggesting S-phase arrest served as a resistance mechanism of cancer cells to spare cytotoxicity of nucleoside analogues. However, nontoxic concentrations of UCN-01 rapidly killed S-phase-arrested cells by apoptosis. Furthermore, the molecular mechanism for UCN-01-induced apoptosis in S-phase-arrested cells was through inhibition of survival pathways associated with these cells. In this regard, suppression of the PI 3-kinase-Akt-Bad survival pathway as well as the NF-κB signaling pathway were associated with induction of apoptosis in S-phase-arrested cells by UCN-01, whereas the Ras-Raf-MEK-ERK pathway appeared not involved. This study has provided the rationales and strategies for optimizing the design of effective combination therapies to overcome resistance to nucleoside analogues. In fact, a clinical trial of the combination of ara-C with UCN-01 to treat relapsed or refractory AML patients has been initiated at U.T.M.D. Anderson Cancer Center. ^

Gene clustering of the small leucine -rich repeat gene family

The small leucine-rich repeat proteoglycans (or SLRPs) are a group of extracellular proteins (ECM) that belong to the leucine-rich repeat (LRR) superfamily of proteins. The LRR is a protein folding motif composed of 20–30 amino acids with leucines in conserved positions. LRR-containing proteins are present in a broad spectrum of organisms and possess diverse cellular functions and localization. In mammals, the SLRPs are abundant in connective tissues, such as bones, cartilage, tendons, skin, and blood vessels. We have discovered a new member of the class I small leucine rich repeat proteoglycan (SLRP) family which is distinct from the other class I SLRPs since it possesses a unique stretch of aspartate residues at its N-terminus. For this reason, we called the molecule asporin. The deduced amino acid sequence is about 50% identical (and 70% similar) to decorin and biglycan. However, asporin does not contain a serine/glycine dipeptide sequence required for the assembly of O-linked glycosaminoglycans and is probably not a proteoglycan. The tissue expression of asporin partially overlaps with the expression of decorin and biglycan. During mouse embryonic development, asporin mRNA expression was detected primarily in the skeleton and other specialized connective tissues; very little asporin message was detected in the major parenchymal organs. The mouse asporin gene structure is similar to that of biglycan and decorin with 8 exons. The asporin gene is localized to human chromosome 9q22-9g21.3 where asporin is part of a SLRP gene cluster that includes ECM2, osteoadherin, and osteoglycin. This gene cluster of four LRR-encoding genes is embedded in a 238 kilobase intron of another novel gene named Tes9orf that is expressed primarily in the testes of the adult mouse. The SLRP genes are not present in Drosophila or C. elegans , but reside in three separate gene clusters in the puffer fish, mice and humans. Targeted disruption of individual mouse SLRP genes display minor connective tissue defects such as skin fragility, tendon laxity, minor growth plate defects, and mild osteoporosis. However, double and triple knockouts of SLRP genes exacerbate these phenotypes. Both the double epiphycan/biglycan and the triple PRELP/fibromodulin/biglycan knockout mice exhibit premature osteoarthritis. ^

Binding of the HIV-1 gp120 -V3 to cellular glycosphingolipids is necessary for CXCR4 recruitment and infection

Infection by human immunodeficiency virus type 1 (HIV-1) is a multi-step process, and detailed analyses of the various events critical for productive infection are necessary to clearly understanding the infection process and identifying novel targets for therapeutic interventions. Evidence from this study reveals binding of the viral envelope protein to host cell glycosphingolipids (GSLs) as a novel event necessary for the orderly progression of the host cell-entry and productive infection by HIV-1. Data obtained from co-immunoprecipitation analyses and confocal microscopy showed that the ability of viral envelope to interact with the co-receptor CXCR4 and productive infection of HIV-1 were inhibited in cells rendered GSL-deficient, while both these activities were restored after reconstitution of the cells with specific GSLs like GM3. Furthermore, evidence was obtained using peptide-inhibitors of HIV-1 infection to show that binding of a specific region within the V3-loop of the envelope protein gp120 to the host cell GSLs is the trigger necessary for the CD4-bound gp120 to recruit the CXCR4 co-receptor. Infection-inhibitory activity of the V3 peptides was compromised in GSL-deficient cells, but could be restored by reconstitution of GSLs. Based on these findings, a revised model for HIV-1 infection is proposed that accounts for the established interactions between the viral envelope and host cell receptors while enumerating the importance of the new findings that fill the gap in the current knowledge of the sequential events for the HIV-1 entry. According to this model, post-CD4 binding of the HIV-1 envelope surface protein gp120 to host cell GSLs, mediated by the gp120-V3 region, enables formation of the gp120-CD4-GSL-CXCR4 immune-complex and productive infection. The identification of cellular GSLs as an additional class of co-factors necessary for HIV-1 infection is important for enhancing the basic knowledge of the HIV-1 entry that can be exploited for developing novel antiviral therapeutic strategies. ^