The effect of protein tyrosine phosphatase SHP1 on tumorigenicity of the MDA -MB231 breast cancer cells

SHP1 is a cytosolic protein tyrosine phosphatase that contains two SH2 domains. It is highly expressed in hematopoietic cells and expressed in normal epithelium at lower levels. While SHP1 in hematopoietic cells is thought to be a negative regulator of cellular signaling by associating with and dephosphorylating various receptors and their downstream effectors after they become activated, its precise function in epithelium remains to be understood. The potential involvement of SHP1 in human tumorigenesis has been hypothesized from the findings that SHP1 can interact with, dephosphorylate, and regulate the activity of several protein tyrosine kinases (PTKs) implicated in human cancer. These PTKs include epidermal growth factor receptor (EGFR) and Src. Such speculation is also supported by the report that SHP1 is overexpressed in human ovarian cancers. ^ Here we report, for the first time, that the levels of SHP1 expression and activity are altered in human breast cancer cells in comparison with normal breast epithelium. In particular, SHP1 expression is nearly lost in the breast cancer cell lines MDA-MB231 and MDA-MB435. After the re-introduction of SHP1 both in wild type (wt) and enzymatically inactive (dn) forms, into the MDA-MB231 cells, we observed no changes in cellular proliferation. However, the overexpression of wt SHP1 led to increased anchorage-independent growth in the MDA-MB231 cells. SHP1 phosphatase activity is essential for such an increase since the overexpression of dn SHP1 had no effect. Enhanced turnorigenicity in nude mice was also observed in the MDA-MB231 cells overexpressing wt SHP1, but not dn SHP1, suggesting the crucial function of SHP1 enzymatic activity in this process. Our observations in this study indicate that SHP1 promotes tumorigenesis by a mechanism or mechanisms apart from enchancing angiogenesis. In addition, we have found no evidence that the overexpression of SHP1 could affect metastatic potential in the MDA-MB231 cells. ^ In the MDA-MB231 cells stably transfected with either wt or dn SHP1 the peak level of EGFR tyrosine phosphorylation induced by EGF, as well as the sensitivity to EGF stimulation, was not altered. However, the overexpression of wt SHP1 led to a slight increase in the kinetics of EGFR dephosphorylation, whereas the overexpression of dn SHP1 led to slightly delayed kinetics of EGFR dephosphorylation. The overexpression of either the wt or dn SHP1 did not lead to any significant increase in Src kinase activity. ^ In NIH3T3 cells, the transient overexpression of SHP1 led to no significant changes in MAP kinase (ERK2) activation by EGF or Akt activation by PDGF. In 3T3H4 cells, the transient overexpression of SHP1 led to no significant changes in MAP kinase (ERK2) activation by heregulin. The transient overexpression of wt SHP1 in the MDA-MB231 cells caused an apparent increase, ranging from 10% to 20%, in the G0/G1 population of the cells with a corresponding decrease in the S phase population. ^ In order to understand the mechanisms by which SHP1 exerts its positive effect on the tumorigenic potential of the MDA-MB231 cells, we employed two-dimensional electrophoresis in an attempt to identify cellular protein(s) with significantly altered tyrosine phosphorylation level upon wt SHP1 overexpression. The overexpression of wt SHP1 but not dn SHP1, leads increased tyrosine phosphorylation of a protein with a molecular weight of approximately 40 kDa and a pI between 5.9 to 6.6. ^

Mechanism of mRNA stability control mediated by AU -rich element: Characterization of cis-elements and trans-factors

Regulation of cytoplasmic deadenylation, the first step in mRNA turnover, has direct impact on the fate of gene expression. AU-rich elements (AREs) found in the 3′ untranslated regions of many labile mRNAs are the most common RNA-destabilizing elements known in mammalian cells. Based on their sequence features and functional properties, AREs can be divided into three classes. Class I or class III ARE directs synchronous deadenylation, whereas class II ARE directs asynchronous deadenylation with the formation of poly(A)-intermediates. Through systematic mutagenesis study, we found that a cluster of five or six copies of AUUUA motifs forming various degrees of reiteration is the key feature dictating the choice between asynchronous versus synchronous deadenylation. A 20–30 nt AU-rich sequence immediately 5 ′ to this cluster of AUUUA motifs can greatly enhance its destabilizing ability and is an integral part of the AREs. These two features are the defining characteristics of class II AREs. ^ To better understand the decay mechanism of AREs, current methods have several limitations. Taking the advantage of tetracycline-regulated promoter, we developed a new transcriptional pulse strategy, Tet-system. By controlling the time and the amount of Tet addition, a pulse of RNA could be generated. Using this new system, we showed that AREs function in both growth- and density-arrested cells. The new strategy offers for the first time an opportunity to investigate control of mRNA deadenylation and decay kinetics in mammalian cells that exhibit physiologically relevant conditions. ^ As a member of heterogeneous nuclear RNA-binding protein, hnRNP D 0/AUF1 displays specific affinities for ARE sequences in vitro . But its in vivo function in ARE-mediated mRNA decay is unclear. AUF1/hnRNP D0 is composed of at least four isoforms derived by alternative RNA splicing. Each isoform exhibits different affinity for ARE sequence in vitro. Here, we examined in vivo effect of AUF1s/hnRNP D0s on degradation of ARE-containing mRNA. Our results showed that all four isoforms exhibit various RNA stabilizing effects in NIH3T3 cells, which are positively correlated with their binding affinities for ARE sequences. Further experiments indicated that AUF1/hnRNP D0 has a general role in modulating the stability of cytoplasmic mRNAs in mammalian cells. ^

A novel DNA damage inducible inhibitor of p53

p53 is required for the maintenance of the genomic stability of cells. Mutations in the p53 tumor-suppressor gene occur in more than 50% of human cancers of diverse types. In addition, 70% of families with Li-Fraumeni syndrome have a germline mutation in p53, predisposing these individuals to multiple forms of cancer. In response to DNA damage, p53 becomes stabilized and activated. However the exact mechanism by which DNA damage signals the stabilization and activation of p53 still remains elusive. The biochemical activity of p53 that is required for tumor suppression, and presumably the cellular response to DNA damage, involves the ability of the protein to bind to specific DNA sequences and to function as a transcription factor. For the downstream targets, p53 transactivates many genes involved in growth arrest, apoptosis and DNA repair such as p21, Bax and GADD45, respectively. An open question in the field is how cells can determine the downstream effects of p53. ^ We hypothesize that, through its associated proteins, p53 can differentially transactivate its target genes, which determine its downstream effect. Additionally, p53 interacting proteins may be involved in signaling for the stabilization and activation of p53. Therefore, a key aspect to understanding p53 function is the identification and analysis of proteins that interact with it. We have employed the Sos recruitment system (SRS), a cytoplasmic yeast two-hybrid screen to identify p53 interacting proteins. The SRS is based on the ability of Sos to activate Ras when it becomes localized to the plasma membrane. The system takes advantage of an S. cerevisiae strain, cdc25-2 temperature sensitive mutant, harboring a mutation in Sos. In this strain, fusion proteins containing a truncated Sos will only localize to the membrane by protein-protein interaction, which allows growth at non-permissive temperature. This system allows the use of intact transcriptional activators such as p53. ^ To date, using a modified SRS library screen to identify p53 interacting proteins, I have identified p53 (known to interact with itself) and a novel p53-interacting protein (PIP). PIP is a specific p53 interacting protein in the SRS. The interaction of p53 and PIP was further confirmed by performing in vitro and in vivo binding assays. In the in vivo binding study, the interaction can only be detected in the presence of ionizing radiation suggesting that this interaction might be involved in DNA-damage induced p53-signalling pathway. After screening cDNA and genomic libraries, a full-length PIP-cDNA clone ( ∼ 3kb) was obtained which encodes a protein of 429 amino acids with calculated molecular weight of 46 kDa. The results of genebank search indicated that the PIP is an unidentified gene and contains a conserved ring-finger domain, which is present in a diverse family of regulatory proteins involved in different aspects of cellular function. Northern blot analysis revealed that the size of its messenge is approximately 3 kb preferentially expressed in brain, heart, liver and kidney. The PIP protein is mainly located in the cytoplasm as determined by the cellular localization of a green fluorescence fusion protein. Preliminary functional analysis revealed that PIP downregulated the transactivation activity of p53 on both p21 and mdm2 promoters. Thus, PIP may be a novel negative regulator of p53 subsequent to DNA damage. ^

A distinct element involved in the lipopolysaccharide activation of the tumor necrosis factor -alpha promoter in a promonocytic cell line, THP-1

TNF-α is a pleiotropic cytokine involved in normal homeostasis and plays a key role in defending the host from infection and malignancy. However when deregulated, TNF-α can lead to various disease states. Therefore, understanding the mechanisms by which TNF-α is regulated may aid in its control. In spite of the knowledge gained regarding the transcriptional regulation of TNF-α further characterization of specific TNF-α promoter elements remains to be elucidated. In particular, the T&barbelow;NF-α A&barbelow;P-1/C&barbelow;RE-like (TAC) element of the TNF-α promoter has been shown to be important in the regulation of TNF-α in lymphocytes. Activating transcription factor-2 (ATF-2) and c-Jun were shown to bind to and transactivate the TAC element However, the role of TAC and transcription factors ATF-2 and c-Jun in the regulation of TNF-α in monocytes is not as well characterized. Lipopolysaccharide (LPS), a potent activator of TNF-α in monocytes, provides a good model to study the involvement of TAC in TNF-α regulation. On the other hand, all-tram retinoic acid (ATRA), a physiological monocyte-differentiation agent, is unable to induce TNF-α protein release. ^ To delineate the functional role of TAC, we transfected the wildtype or the TAC deleted TNF-α promoter-CAT construct into THP-1 promonocytic cells before stimulating them with LPS. CAT activity was induced 17-fold with the wildtype TNF-α promoter, whereas the CAT activity was uninducible when the TAC deletion mutant was used. This daft suggests that TAC is vital for LPS to activate the TNF-α promoter. Electrophoretic mobility shift assays using the TAC element as a probe showed a unique pattern for LPS-activated cells: the disappearance of the upper band of a doublet seen in untreated and ATRA treated cells. Supershift analysis identified c-Jun and ATF-2 as components of the LPS-stimulated binding complex. Transient transfection studies using dominant negative mutants of JNK, c-Jun, or ATF-2 suggest that these proteins we important for LPS to activate the TNF-α promoter. Furthermore, an increase in phosphorylated or activated c-Jun was bound to the TAC element in LPS-stimulated cells. Increased c-Jun activation was correlated with increased activity of Jun N-terminal kinase (JNK), a known upstream stimulator of c-Jun and ATF-2, in LPS-stimulated monocytes. On the other hand, ATRA did not induce TNF-α protein release nor changes in the phosphorylation of c-Jun or JNK activity, suggesting that pathways leading to ATRA differentiation of monocytic cells are independent of TNF-α activation. Together, the induction of TNF-α gene expression seems to require JNK activation, and activated c-Jun binding to the TAC element of the TNF-α promoter in THP-1 promonocytic cells. ^

Characterization of anti -sulfatide autoantibodies in demyelinating diseases

Multiple sclerosis (MS) is the most common autoimmune disease of the central nerve system and Guillain Barré Syndrome (GBS) is an inflammatory neuropathy involving the peripheral nerves. Anti-myelin immunoglobins may play a role in the demyelination processes of the both diseases. Sulfatide is an abundant glycolipid on myelin and is a candidate target antigen for disease related autoantibodies. The objective of this study was to characterize anti-sulfatide antibodies and compare antibodies from GBS and MS patients with fetal antibodies. Our hypothesis is that some B cells producing disease-associated autoantibodies are derived from or related to B cells of the fetal repertoire. Here we report that reactivity of plasma IgM against sulfatide was elevated in twelve MS patients compared with twelve normal subjects. This result implies that anti-sulfatide antibodies are disease-related. A total of sixteen human B lymphocyte clones producing anti-sulfatide autoantibodies were isolated from MS patients, GBS patients and a human fetus. Seven of the clones were from three MS patients, four of the clones were from three GBS patients and five were from the spleen of a twenty-week human fetus. Sequences have been obtained for the heavy and light chain variable regions (VDJ and VJ regions) of all of the anti-sulfatide immunoglobulins. Seven of the sixteen antibodies used VH3 for the variable region gene of the heavy chain consistent with the rate of VH3 usage in randomly selected B cells. Somatic mutations were significantly more frequent in the patient antibodies than in the fetus and somatic mutations in CDR's (Complementarity Determining Region) were significantly more frequent than in framework regions. No significant difference was found between patients and fetus in length of VH CDRIII. However, it is reported that antibodies from randomly selected normal adult B cells have longer CDRIII lengths than those of the fetus (Sanz I, 1991 Journal of Immunology Sep 1;147(5):1720-9). Our results are consistent with derivation of the precursors of B cells producing these autoantibodies from B cells related to those of the fetal repertoire. These findings are consistent with a model in which quiescent B cells from clones produced early in development undergo proliferation in dysregulated disease states, accumulating somatic mutations and increasing in reactivity toward self-antigens. ^ Epitope mapping and molecular modeling were done to elucidate the relationships between antibody structure and binding characteristics. The autoantibodies were tested for binding activity to three different antigens: sulfatide, galactoceramide and ceramide. Molecular modeling suggests that antibodies with positive charge surrounded by or adjacent to hydrophobic groups in the binding pocket bind to the head of sulfatide via the sulfate group through electrostatic interactions. However, the antibodies with hydrophobic groups separated from positive charges appear to bind to the hydrophobic tail of sulfatide. This observation was supported by a study of the effect of NaCl concentration on antigen binding. The result suggested that electrostatic interactions played a major role in sulfate group binding and that hydrophobic interactions were of greater importance for binding to the ceramide group. Our three-dimensional structure data indicated that epitope specificity of these antibodies is more predictable at the level of tertiary than primary structure and suggested positive selection based on structure occurred in the. formation of those autoantibodies. ^

A novel mechanism of regulation of phosphatidylinositol 3 -kinase: Tyrosine phosphorylation of p85 residue 688

Phosphatidylinositol 3-kinase (PI3K) phosphorylates membrane constituent phosphatidylinositols, producing second messengers that link membrane bound receptor signals to cellular proliferation and survival. PI3K, a heterodimer consisting of a catalytic p110 subunit and a regulatory p85 subunit, can be activated through induced association with other signaling molecules. The p85 subunit serves to both stabilize and inactivate p110. The inhibitory activity of P85 is relieved by occupancy of the N terminal SH2 domain by phosphorylated tyrosine. PI3K becomes phosphorylated and activated subsequent to a variety of stimuli. Indeed, Src family kinases have been demonstrated to phosphorylate p85 at tyrosine 688, but the role of phosphorylation in PI3K function is unclear. We decided to evaluate the importance of tyrosine phosphorylation to PI3K activity. We demonstrate that tyrosine phosphorylated p85 is associated with a higher specific activity than is non-phosphorylated PI3K. Wild type p85 inhibits PI3K enzyme activity, a process accentuated by mutation of tyrosine 688 to alanine and reversed by mutation to aspartate which functions as a phosphotyrosine mimic in multiple systems. Strikingly, the Y688D mutation completely reverses the p85 inhibitory activity on cell viability and activation of downstream protein NFkB. We demonstrate that tyrosine phosphorylated Y688 or Y688D is sufficient to bind the p85 N terminal SH2 domain, either within full length p85 or in an isolated N terminal SH2 domain, suggesting the possibility of an intramolecular interaction between phosphorylated Y688 and the p85 N terminal SH2 domain that can relieve the p85-induced inhibition of p110. Further, we provide evidence that dephosphorylation of Y688 reduces phosphorylation-induced PI3K activity. We demonstrate that tyrosine phosphatase SHP-1 can physically associate with p85 in a SH2-mediated interaction with the C terminal tail of SHP-1. This association is concomitant with both p85 dephosphorylation and decreased PI3K activity. Altogether, our data suggests the phosphorylation state of p85 is the focal point of a novel mechanism for PI3K activity regulation. As PI3K has been shown to be involved in the vital physiological processes of cell proliferation and apoptosis, a thorough understanding of the regulation of this signaling protein may provide opportunities for the design of novel treatments for cancer. ^

Role of janus tyrosine kinase -3 (JAK3) and signal transducer and activator of transcription (STAT5) pathway in T lymphocyte activation

T cell activation and expansion is essential for immune response against foreign antigens. However, uncontrolled T cell activity can be manifested as a number of lymphoid derived diseases such as autoimmunity, graft versus host disease, and lymphoma. The purpose of this research was to test the central hypothesis that the Jak3/Stat5 pathway is critical for T cell function. To accomplish this objective, two novel Jak3 inhibitors, AG490 and PNU156804, were identified and their effects characterized on Jak3/Stat5 activation and T cell growth. Inhibition of Jak3 selectively disrupted primary human T lymphocyte growth in response to Interleukin-2 (IL-2), as well as other γ c cytokine family members including IL-4, IL-7, IL-9, and IL-15. Inhibition of Jak3 ablated IL-2 induced Stat5 but not TNF-α mediated NF-κβ DNA binding. Loss of Jak3 activity did not affect T cell receptor mediated signals including activation of p56Lck and Zap70, or IL-2 receptor a chain expression. To examine the effects of Jak3/Stat5 inhibition within a mature immune system, we employed a rat heart allograft model of Lewis (RT1 1) to ACI (RT1a). Heart allograft survival was significantly prolonged following Jak3/Stat5 inhibition when rats were treated with AG490 (20mg/kg) or PNU156804 (80mg/kg) compared to non-treated control animals. This effect was synergistically potentiated when Jak3 inhibitors were used in combination with a signal 1/2 disrupter, cyclosporine, but only additively potentiated with another signal 3 inhibitor, rapamycin. This suggested that sequential inhibition of T cell function is more effective. To specifically address the role of Stat5 in maintaining T cell activity, novel Stat5 antisense oligonucleotides were synthesized and characterized in vitro. Primary human T cells and T-cell tumor lines treated with Stat5 antisense oligonucleotide (7.5 μM) rapidly underwent apoptosis, while no changes in cell cycle were observed as measured by FACS analysis utilizing Annexin-V-Fluorescein and Propidium iodide staining. Evidence is provided to suggest that caspase 8 and 9 pathways mediate this event. Thus, Stat5 may act rather as a negative regulator of apoptotic signals and not as a positive regulator of cell cycle as previously proposed. We conclude that the Jak3/Stat5 pathway is critical for γc cytokine mediated gene expression necessary for T cell expansion and normal immune function and represents an therapeutically relevant effector pathway to combat T cell derived disease. ^

Regulation of Bcl-2 expression by the product of the prostate apoptosis response gene -4, Par-4

Elevated expression levels of the bcl-2 proto-oncogene have been correlated with the appearance of androgen independence in prostate cancer. Although bcl-2 was first cloned as the t (14:18) translocation breakpoint from human follicular B cell lymphoma, the mechanism of overexpression of bcl-2 is largely undefined for advanced prostate cancer, there being no gross alterations in the gene structure. We investigated the role of the product of the prostate apoptosis response gene-4 (Par-4) and the product of the Wilms' tumor 1 gene (WT1) in the regulation of Bcl-2 expression in prostate cancer cell lines. We observed growth arrest and apoptosis, upon decreasing Bcl-2 protein and transcript in the high Bcl-2 expressing, androgen-independent prostate cancer cell lines, by all trans-retinoic acid treatment but this did not occur in the androgen-dependent cell lines expressing low levels of Bcl-2. Changes in localization of Par-4, and an induction in the expression of WT1 protein accompanied the decrease in the Bcl-2 protein and transcript following all trans-retinoic acid treatment, in the androgen-independent prostate cancer cell line. In stable clones expressing ectopic Par-4 we observed decreased Bcl-2 protein and transcript. This was accompanied by an induction in WT1 expression. Finally, we detected Par-4 and WT1 proteins binding to a previously identified WT1 binding site on the bcl-2 promoter both in vitro and in vivo leading to a decrease in transcription from the bcl-2 promoter. We conclude that Par-4 regulates Bcl-2 through a WT1 binding site on the bcl-2 promoter. ^

Impact of BCR -ABL on DNA repair

The BCR-ABL fusion gene is the molecular hallmark of Philadelphia-positive leukemias. Normal Bcr is a multifunctional protein, originally localized to the cytoplasm. It has serine kinase activity and has been implicated in cellular signal transduction. Recently, it has been reported that Bcr can interact with xeroderma pigmentosum group B (XPB/ERCC3)—a nuclear protein active in UV-induced DNA repair. Two major Bcr proteins (p160 Bcr and p130Bcr) have been characterized, and our preliminary results using metabolic labeling and immunoblotting demonstrated that, while both the p160 and p130 forms of Bcr localized to the cytoplasm, the p130 form (and to a lesser extent p160) could also be found in the nucleus. Furthermore, electron microscopy confirmed the presence of Bcr in the nucleus and demonstrated that this protein associates with metaphase chromatin as well as condensed interphase heterochromatin. Since serine kinases that associate with condensed DNA are often cell cycle regulatory, these observations suggested a novel role for nuclear Bcr in cell cycle regulation and/or DNA repair. However, cell cycle synchronization analysis did not demonstrate changes in levels of Bcr throughout the cell cycle. Therefore we hypothesized that BCR serves as a DNA repair gene, and its function is altered by formation of BCR-ABL. This hypothesis was investigated using cell lines stably transfected with the BCR-ABL gene, and their parental counterparts (MBA-1 vs. M07E and Bcr-AblT1 vs. 4A2+pZAP), and several DNA repair assays: the Comet assay, a radioinimunoassay for UV-induced cyclobutane pyrimidine dimers (CPDs), and clonogenic assays. Comet assays demonstrated that, after exposure to either ultraviolet (UV)-C (0.5 to 10.0 joules m −2) or to gamma radiation (200–1000 rads) there was greater efficiency of DNA repair in the BCR-ABL-transfected cells compared to their parental controls. Furthermore, after UVC-irradiation, there was less production of CPDs, and a more rapid disappearance of these adducts in BCR-ABL-bearing cells. UV survival, as reflected by clonogenic assays, was also greater in the BCR-ABL-transfected cells. Taken together, these results indicate that, in our systems, BCR-ABL confers resistance to UVC-induced damage in cells, and increases DNA repair efficiency in response to both UVC- and gamma-irradiation. ^

Structural and functional analysis of p84N5

Normal development and tissue homeostasis requires the carefully orchestrated balance between cell proliferation and cell death. Cell cycle checkpoints control the extent of cell proliferation. Cell death is coordinated through the activation of a cell suicide pathway that results in the morphologically recognizable form of death, apoptosis. Tumorigenesis requires that the balance between these two pathways be disrupted. The tumor suppressor protein Rb has not only been shown to be involved in the enforcement of cell cycle checkpoints, but has also been implicated in playing a role in the regulation of apoptosis. The manner in which Rb enforces cell cycle checkpoints has been well studied; however, its involvement in the regulation of apoptosis is still very unclear. p84N5 is a novel nuclear death domain containing protein that has been shown to interact with the N-terminus of Rb. The fact that it contains a death domain and the fact that it is nuclear localized possibly provides the first known mechanism for apoptotic signaling from the nucleus. The following study tested the hypothesis that the novel exclusively nuclear death domain containing protein p84N5 is an important mediator of programmed cell death and that its apoptotic function is reliant upon its nuclear localization and is regulated by unique functional domains within the p84N5 protein. We identified the p84N5 nuclear localization signal (NLS), eliminated it, and tested the functional significance of nuclear localization by using wild type and mutant sequences fused to EGFP-C1 (Clontech) to create wild type GFPN5 and subsequent mutants. The results of these assays demonstrated exclusive nuclear localization of GFPN5 is required for normal p84N5 induced apoptosis. We further conducted large-scale mutagenesis of the GFPN5 construct to identify a minimal region within p84N5 capable of interacting with Rb. We were able to identify a minimal sequence containing p84N5 amino acids 318 to 464 that was capable of interacting with Rb in co-immunoprecipitation assays. We continued by conducting a structural and functional analysis to identify the region or regions within p84N5 responsible for inducing apoptosis. Point mutations and small-scale deletions within the death domain of p84N5 lessened the effect but did not eliminate p84N5-induced cytotoxicity. Further analysis revealed that the minimal sequence of 318 to 464 of p84N5 was capable of inducing apoptosis to a similar degree as wild-type GFPN5 protein. Since amino acids 318 to 464 of p84N5 are capable of inducing apoptosis and interacting with Rb, we propose possible mechanisms whereby p84N5 may function in a Rb regulated manner. These results demonstrate that p84N5 induced apoptosis is reliant upon its nuclear localization and is regulated by unique functional domains within the p84N5 protein. ^

A role for p53 in sensing DNA damage and triggering apoptotic responses to anticancer agents

The p53 tumor suppressor protein plays a major role in cellular responses to anticancer agents that target DNA. DNA damage triggers the accumulation of p53, resulting in the transactivation of genes, which induce cell cycle arrest to allow for repair of the damaged DNA, or signal apoptosis. The exact role that p53 plays in sensing DNA damage and the functional consequences remain to be investigated. The main goal of this project was to determine if p53 is directly involved in sensing DNA damage induced by anticancer agents and in mediating down-stream cellular responses. This was tested in two experimental models of DNA damage: (1) DNA strand termination caused by anticancer nucleoside analogs and (2) oxidative DNA damage induced by reactive oxygen species (ROS). Mobility shift assays demonstrated that p53 and DNA-PK/Ku form a complex that binds DNA containing the anticancer nucleoside analog gemcitabine monophosphate in vitro. Binding of the p53-DNA-PK/Ku complex to the analog-containing DNA inhibited DNA strand elongation. Furthermore, treatment of cells with gemcitabine resulted in the induction of apoptosis, which was associated with the accumulation of p53 protein, its phosphorylation, and nuclear localization, suggesting the activation of p53 to trigger apoptosis following gemcitabine induced DNA strand termination. The role of p53 as a DNA damage sensor was further demonstrated in response to oxidative DNA damage. Protein pull-down assays demonstrated that p53 complexes with OGG1 and APE, and binds DNA containing the oxidized DNA base 8-oxoG. Importantly, p53 enhances the activities of APE and OGG1 in excising the 8-oxoG residue as shown by functional assays in vitro. This correlated with the more rapid removal of 8-oxoG from DNA in intact cells with wild-type p53 exposed to exogenous ROS stress. Interestingly, persistent exposure to ROS resulted in the accelerated onset of apoptosis in cells with wild-type p53 when compared to isogenic cells lacking p53. Apoptosis in p53+/+ cells was associated with accumulation and phosphorylation of p53 and its nuclear localization. Taken together, these results indicate that p53 plays a key role in sensing DNA damage induced by anticancer nucleoside analogs and ROS, and in triggering down-stream apoptotic responses. This study provides new mechanistic insights into the functions of p53 in cellular responses to anticancer agents. ^

Upregulation of VEGF through p70S6K in ErbB2 -mediated angiogenesis and inhibition by combined herceptin plus taxol therapy

ErbB2 overexpression in breast tumors increases metastasis, angiogenesis, and reduces survival. To study ErbB2 signaling mechanisms in metastasis and angiogenesis, a spontaneous metastasis assay was performed using human breast cancer cells transfected with constitutively active ErbB2 kinase (V659E), an ErbB2 kinase-dead mutant (K753M), or vector control. Mice injected with V659E had increased metastasis and tumor microvessel density; and the increased angiogenesis in vivo from the V659E transfectants paralleled increased angiogenic potential in vitro, which resulted from increased VEGF by increased protein synthesis. This appeared to be mediated through a PI3K, Akt, mTOR, p70S6K-signaling pathway. Furthermore, V659E xenografts had significantly increased phosphorylated Akt, phosphorylated p70S6K, and VEGF compared with control. To validate the clinical relevance of these findings, human breast tumor samples were examined. Tumors overexpressing ErbB2 correlated with p70S6K phosphorylation and VEGF expression, which significantly correlated with higher levels of Akt and mTOR phosphorylation. Additionally, patients with tumors having increased p70S6K phosphorylation showed a trend for worse disease-free survival and increased metastasis. Together, ErbB2 increases VEGF expression by activating the p70S6K signaling pathway, which may serve as targets for antiangiogenic and antimetastatic therapies. ^ Herceptin is an anti-ErbB2 antibody that demonstrated anti-tumor function, especially in combination with other chemotherapies such as Taxol, in patients with ErbB2-overexpressing tumors. Since the repeated administration of low-dose chemotherapy endorsed an antiangiogenic effect in vitro, and Herceptin was shown to inhibit angiogenesis in tumor xenografts, I investigated whether combined Taxol plus Herceptin treatment inhibits ErbB2-mediated angiogenic responses more effectively. Mice with ErbB2-overexpressing xenografts were treated with control, Herceptin, Taxol, or combination Herceptin plus Taxol. Mice treated with the combination exhibited reduced tumor volumes, tumor microvessel densities, and lung metastasis; and ErbB2-overexpressing cells treated with the combination secreted less VEGF, and stimulated less endothelial cell migration. Furthermore, Akt phosphorylation contributed to VEGF upregulation and was most effectively reduced by combination treatment. ^ In summary, ErbB2 activates signaling to Akt and p70S6K leading to increased VEGF and angiogenesis. Combination Herceptin plus Taxol treatment most effectively inhibited ErbB2-mediated angiogenesis, resulting in pronounced tumoricidal effects, and may be mediated through reduction of phosphorylated Akt, a positive regulator in the p70S6K pathway. ^

Molecular mechanism of the relaxin signaling pathways: Activation of cAMP, PI3K, and PKCzeta

Relaxin is a polypeptide hormone that has diverse effects on reproductive and non-reproductive tissues. Relaxin activates the G-protein coupled receptors, LGR7 and LRG8. Early studies described increased cAMP and protein kinase A activity upon relaxin treatment, but cAMP accumulation alone could not account for all of the relaxin-mediated effects. We utilized the human monocyte cell line THP-1 to study the mechanism of relaxin-stimulated CAMP production. ^ Relaxin treatment in THP-1 cells produces a biphasic time course in cAMP accumulation, where the first peak appears as early as 1–2 minutes with a second peak at 10–20 minutes. Selective inhibitors for phosphoinositide 3-kinase (P13K), such as wortmannin and LY294002, show a dose-dependent inhibition of relaxin-stimulated cAMP accumulation, specific for the second peak of the relaxin time course. Neither the effects of relaxin nor the inhibition of relaxin by LY294002 is mediated by the activity of phosphodiesterases. Furthermore, LY294002 blocks upregulation of vascular endothelial growth factor transcript levels by relaxin. ^ To further delineate relaxin signaling pathways, we searched for downstream targets of PI3K that could activate adenylyl cyclase (AC). Protein kinase C ζ (PKCζ) was a prime candidate because it activates types II and V AC. Chelerythrine chloride (a general PKC inhibitor) inhibits relaxin-induced cAMP production to the same degree as LY294002 (∼40%). Relaxin stimulates PKCζ translocation to the plasma membrane in THP-1, MCF-7, PHM1-31, and MMC cells, as shown by immunocytochemistry. PKCζ translocation is P13K-dependent and independent of cAMP production. Antisense PKCζ oligodeoxynucleotides (PKCζ-ODNs) deplete both PKCζ transcript and protein levels in THP-1 cells. PKCζ-ODNs abolish relaxin-mediated PKCζ translocation and inhibit relaxin stimulation of cAMP by 40%, as compared to mock and random ODN controls. Treatment with LY294002 in the presence of PKCζ-ODNs results in little further inhibition. Taken together, we present a novel role for PI3K and PKCζ in relaxin stimulation of cAMP and provide the first example of the PKCζ regulation of AC in an endogenous system. Furthermore, we have identified higher order complexes of AC isoforms and PKA anchoring proteins in attempts to explain the differential coupling of relaxin to cAMP and PI3K-signaling pathways in various cell types. ^

Genomic variation of HIV-1 and its effects on drug resistance and molecular epidemiology analyses

In the first part of this study human immunodeficiency virus type 1 (HIV-1) proviral DNA sequences derived from 201 clones of the C2-V3 env region and the first exon of the tat gene were obtained from six MV-1 infected heterosexual couples. These molecular data were used to confirm the epidemiological relationships. The ability of the molecular data to draw such conclusions was also tested with multiple phylogenetic analyses. The tat region was much more useful in establishing epidemiological relationships than the commonly used C2-V3.^ Subsequently, using nucleotide sequences from the first exon of the Tat gene, we tested the hypothesis that a Florida dentist (a common source) infected five of his patients in the course of dental procedures, against the null hypothesis that the dentist and each individual of the dental group independently acquired the virus within the local community. Multiple phylogenetic analyses demonstrated that the sequences of the five patients were significantly more related to each other than to sequences of the controls. Our results using Tat sequences, combined with envelope sequence data, strongly support a common phylogenetic epidemiological relationship among these five patients.^ A third study is presented, which deals with the effects of genomic variations in drug resistance. HIV-1 reverse transcriptase (RT) mutations were detected in DNA from peripheral blood mononuclear cells from 11 of 12 HIV-infected children after 11-20 months of zidovudine monotherapy. The codon 41/215 mutant combination was associated with general decline in health status. Patients developing the codon 70 mutation tended to have a better health status. ^

Human biology and health studies.

Proporciona a los estudiantes los temas requeridos para el primer año del curso GCSE, especialidad biología. Es una edición revisada para el nuevo AQA GCSE en Fisiología humana y estudios de la salud.