Stress-induced targeting of molecular chaperones in the yeast Saccharomyces cerevisiae

The eukaryotic stress response is an essential mechanism that helps protect cells from a variety of environmental stresses. Cell death can result if cells are not able to properly adapt and protect themselves against adverse stress conditions. Failure to properly deal with stress has implications in human diseases including neurodegenerative disorders and distinct cancers, emphasizing the importance of understanding the eukaryotic stress response in detail. As part of this response, expression of a battery of heat shock proteins (HSP) is induced, which act as molecular chaperones to assist in the repair or triage of unfolded proteins. The 90-kDa HSP (Hsp90) operates in the context of a multi-chaperone complex to promote the maturation of nuclear and cytoplasmic clients. I have discovered that Hsp90 and the co-chaperone Sba1 accumulate in the nucleus of quiescent Saccharomyces cerevisiae cells in a karyopherin-dependent manner. I isolated nuclear accumulation- defective HSP82 mutant alleles to probe the nature of this targeting event and identified a mutant with a single amino acid substitution (I578F) sufficient to prevent nuclear accumulation of Hsp90 in quiescent cells. Diploid hsp82-I578F cells exhibited pronounced defects in spore wall construction and maturation, resulting in catastrophic sporulation. The mislocalization and sporulation phenotypes were shared by another previously identified HSP82 mutant allele, further linking localization to Hsp90 functional status. Pharmacological inhibition of Hsp90 with macbecin in sporulating diploid cells also blocked spore formation, underscoring the importance of this chaperone in this developmental program. The yeast molecular chaperone Hsp104 is a member of the Hsp100 superfamily of AAA+ ATPases. Unlike the Hsp90 family of chaperones, Hsp104 is not restricted to a specific set of client proteins, but rather assists in reactivating stress-denatured proteins by solubilizing protein aggregates. I have discovered that Hsp104, along with the Hsp70 chaperone, Ssa1, and the sHSP Hsp26 accumulate into RNA processing bodies (P- bodies) and stress granules, sites of mRNA metabolism. I found that Hsp104 recruits both Ssa1 and Hsp26 to P-bodies and that these three chaperones are required for stress granule formation. These findings suggest a possible role for chaperones in mRNA metabolism by aiding in the assembly, disassembly or conversion of these enigmatic mRNP complexes. Taken together, the work presented in this dissertation serves to better understand the eukaryotic stress response by illustrating the importance of subcellular-chaperone localization in key biological processes.

Mechanism of surgical stress impairment of murine natural killer cell cytotoxicity

Natural killer cells may provide an important first line of defense against metastatic implantation of solid tumors. This antitumor function occurs during the intravascular and visceral lodgment phase of cancer dissemination, as demonstrated in small animal metastasis models. The role of the NK cell in controlling human tumor dissemination is more difficult to confirm, at least partially because of ethical restraints on experimental design. Nonetheless, a large number of solid tumor patient studies have demonstrated NK cell cytolysis of both autologous and allogeneic tumors.^ Of the major cancer therapeutic modalities, successful surgery in conjunction with other treatments offers the best possibility of cure. However, small animal experiments have demonstrated that surgical stress can lead to increased rates of primary tumor take, and increased incidence, size, and rapidity of metastasis development. Because the physiologic impact of surgical stress can also markedly impair perioperative antitumor immune function in humans, we examined the effect of surgical stress on perioperative NK cell cytolytic function in a murine preclinical model. Our studies demonstrated that hindlimb amputation led to a marked impairment of postoperative NK cell cytotoxicity. The mechanism underlying this process is complex and involves the postsurgical generation of splenic erythroblasts that successfully compete with NK cells for tumor target binding sites; NK cell-directed suppressor cell populations; and a direct impairment of NK cell recycling capacity. The observed postoperative NK cell suppression could be prevented by in vivo administration of pyrimidinone biologic response modifiers or by short term in vitro exposure of effector cells to recombinant Interleukin-2. It is hoped that insights gained from this research may help in the future development of NK cell specific perioperative immunotherapy relevant to the solid tumor patients undergoing cancer resection. ^

Integrins in human T cell function: Transdominant regulation, conformational constraints, and intracellular effectors

Integrin adhesion molecules have both positive and negative potential in the regulation of peripheral blood T cell (PB T cell) activation, yet their mechanism of action in the mediation of human T lymphocyte function remains largely undefined. The goals of this study then were to elucidate integrin signaling mechanisms in PB T cells.^ By ligating $\beta$1 integrins with mAb 18D3, it was demonstrated that costimulation of PB T cell proliferation induced by coimmobilizing antibodies specific for $\beta$1, $\beta$2, and $\beta$7 integrin subfamilies in conjunction with the anti-CD3 mAb OKT3 was inhibited. Costimulation of T cell proliferation induced by non-integrins CD4, CD26, CD28, CD44, CD45RA, or CD45RO was unaffected. Inhibition of costimulation correlated with diminished IL-2 production. In his manner, $\beta$1 integrins could regulate heterologous integrins of the $\beta$2 and $\beta$7 subfamilies in a transdominant fashion. It was also demonstrated that integrin costimulation of T cell activation was acutely sensitive to the structural conformation of $\beta$1 integrins. Using the cyclic hexapeptide CWLDVC (TBC772, which is based on the $\alpha4\beta1$ integrin binding site in fibronectin) in soluble form, it was shown that integrins locked into a conformation displaying a neo-epitope called the ligand induced binding site (LIBS) recognized by mAb 15/7 were inhibited from sending mitogenic signals to T cells. When BSA-conjugated TBC772 was coimmobilized with anti-CD3 mAb OKT3, costimulation of proliferation occurred. This suggested that temporally uncoupling integrin receptor occupancy from receptor crosslinking inhibited $\beta$1 integrin signaling mechanisms. When subsets of PB T cells were examined to determine those initially activated by integrins within 6 hours of activation, costimulation induced intracellular accumulation of IL-2 predominantly in the CD4$\sp+$ and CD45RO$\sp+$ T cell subsets. This was similar to a number of PB T cell costimulatory molecules including CD26, CD43, CD44. Only CD28 costimulated IL-2 production from both CD45RA$\sp+$ and CD45RO$\sp+$ subpopulations.^ The GTPase Rho has been implicated in regulating integrin mediated stress fiber formation and anchorage dependent growth in fibroblasts, so studies were initiated to determine if Rho played a role in integrin dependent T cell function. In order to perform this, a technique based on scrape-loading was developed to incorporate macromolecules into PB T cells that maintained their functional activity. With this technique, C3 exoenzyme from Clostridium botulinum was incorporated into PB T cells. C3 ADP-ribosylates Rho proteins on Asn$\sp{41},$ which is in close proximity to the Rho effector domain, rendering it inactive. It was demonstrated that functional Rho is not required for basal or upregulated PB T cell adhesion to $\beta$1 integrin substrates, however PB T cell homotypic aggregation induced by PMA, which is an event mediated predominantly by the integrin $\rm\alpha L\beta2,$ was delayed. PB T cells lacking Rho function displayed altered cell morphology on $\beta$1 integrin ligands, producing stellate, dendritic-like pseudopodia. Rho activity was also found to be required for integrin dependent costimulation of proliferation. When intracellular accumulation of IL-2 was measured, inactivation of Rho prevented both integrin and CD28 costimulatory activity. Rho was identified to lie upstream of signals mediating PKC activation and Ca$\sp{++}$ fluxes, as PMA and ionomycin activation of PB T cells was unaffected by the inactivation of Rho. ^

REGULATION OF BRAIN NORADRENERGIC-RECEPTOR FUNCTION BY ADRENOCORTICOTROPHIN AND ANTIDEPRESSANT DRUGS (ADRENERGIC RECEPTOR, CYCLIC-AMP, ADENYLATE CYCLASE, IMIPRAMINE, STRESS)

Human behavior appears to be regulated in part by noradrenergic transmission since antidepressant drugs modify the number and function of (beta)-adrenergic receptors in the central nervous system. Affective illness is also known to be associated with the endocrine system, particularly the hypothalamic-pituitary-adrenal axis. The aim of the present study was to determine whether hormones, in particular adrencorticotrophin (ACTH) and corticosterone, may influence behavior by regulating brain noradrenergic receptor function.^ Chronic treatment with ACTH accelerated the increase or decrease in rat brain (beta)-adrenergic receptor number induced by a lesion of the dorsal noradrenergic bundle or treatment with the antidepressant imipramine. Chronic administration of ACTH alone had no effect on (beta)-receptor number although it reduced norepinephrine stimulated cyclic AMP accumulation in brain slices. Treatment with imipramine also reduced the cyclic AMP response to norepinephrine but was accompanied by a decrease in (beta)-adrenergic receptor number. Both the imipramine and ACTH treatments reduced the affinity of (beta)-adrenergic receptors for norepinephrine, but only the antidepressant modified the potency of the neurotransmitter to stimulate second messenger production. Neither ACTH nor imipramine treatment altered Gpp(NH)p- or fluoride-stimulated adenylate cyclase, cyclic AMP, cyclic GMP, or cyclic GMP-stimulated cyclic AMP phosphodiesterase, or the activity of the guanine nucleotide binding protein (Gs). These findings suggested that post-receptor components of the cyclic nucleotide generating system are not influenced by the hormone or antidepressant. This conclusion was verified by the finding that neither treatment altered adenosine-stimulated cyclic AMP accumulation in brain tissue.^ A detailed examination of the (alpha)- and (beta)-adrenergic receptor components of norepinephrine-stimulated cyclic AMP production revealed that ACTH, but not imipramine, administration reduced the contribution of the (alpha)-receptor mediated response. Like ACTH treatment, corticosterone diminished the (alpha)-adrenergic component indicating that adrenal steroids probably mediate the neurochemical responses to ACTH administration. The data indicate that adrenal steroids and antidepressants decrease noradrenergic receptor function by selectively modifying the (alpha)- and (beta)-receptor components. The functional similarity in the action of the steroid and antidepressants suggests that adrenal hormones normally contribute to the maintenance of receptor systems which regulate affective behavior in man. ^

Glucocorticoid alterations in the human type-1/type-2 cytokine balance and the role of CD28/B7 costimulation

We have recently reported that psychological stress is associated with a shift in the human type-1/type-2 cytokine balance toward a type-2 cytokine response. The mechanisms of these cytokine alterations are unknown, but likely involve glucocorticoid (GC) modulation of cytokine production. Therefore we sought to characterize the effects of GC on the in vitro human type-1/type-2 cytokine balance. We hypothesized that GC induce a type-2 cytokine shift through modulation of critical regulatory cytokines and alterations in the CD28/B7 costimulatory pathway. ^ We first sought to characterize the effect of the GC, dexamethasone (DEX), on type-1 (IFN-γ, IL-12) and type-2 (IL-4, IL-10) cytokine production by human peripheral blood mononuclear blood cells (pBMC) stimulated with a variety of T-lymphocyte and monocyte stimuli. DEX, at concentrations mimicking stress and supraphysiologic levels of cortisol, decreased IFN-γ and IL-12 production and increased IL-4 and IL-10 production, indicating a shift in the type-1/type-2 cytokine balance toward a type-2 response. Furthermore, both CD4+ and CD8+ T-lymphocytes were susceptible to the cytokine modulating effects of DEX. Furthermore, in the absence of the monocyte, the DEX-induced alterations in T-lymphocyte cytokine production were reduced, indicating that the interaction between the monocyte and T-lymphocyte plays a significant role. ^ We next determined the role of regulatory cytokines, known to modulate the type-1/type-2 cytokine balance, in the DEX-induced cytokine alterations. The addition of the recombinant IL-12p70 and IFN-γ, but not the neutralization of IL-4, IL-10 or IL-13 using monoclonal antibodies, attenuated the DEX-induced type-1/type-2 cytokine alterations. These data suggest that the DEX-induced cytokine alterations are mediated, at least in part, through the initial inhibition type-1 cytokines. Lastly, we investigated the role of the CD28/B7 costimulatory pathway in these cytokine alterations. DEX decreased the expression of CD80 and CD86 on THP-1 cells, a monocyte cell line, and the expression of CD28 and CTLA-4 on PHA-stimulated pBMC. The DEX-induced decrease in CD28 and CTLA-4 expression was attenuated by rhIL-12. Finally, CD28 activation attenuated the DEX-induced decrease in IFN-γ production, suggesting that modulation of the CD28/B7 costimulatory pathway may contribute to the DEX-induced type-1/type-2 cytokine alterations. ^

Mammalian Snm1 participates in cellular responses to genotoxic and mitotic stress

Eukaryotic cells have evolved a complex network of metabolic processes and regulatory systems to help ensure that hereditary information is protected or restored when exposed to genotoxic agents. Two members of the Snm1 protein family have been characterized; scSNM1/PSO2, a yeast gene responsible for repair of DNA interstrand crosslinks, and hARTEMIS, a human gene that is mutated in radiosensitive severe combined immunodeficiency (RS-SCID). Here we report on another member of this protein family, hSNM1, and its response to DNA damage and mitotic stress. We have found that this protein colocalizes and physically associates with 53BP1, a crucial member of the mammalian response to DNA damage. In addition, hSnm1 interacts with several proteins involved in mitosis, and mSNM1 deficiency causes a mitotic checkpoint defect in mouse embryonic fibroblasts. ^

14-3-3zeta overexpression in multiple human cancers plays a critical role in cancer progression

14-3-3 is a family of highly conserved and ubiquitously expressed proteins in eukaryotic organisms. 14-3-3 isoforms bind in a phospho-serine/threonine-dependent manner to a host of proteins involved in essential cellular processes including cell cycle, signal transduction and apoptosis. We fortuitously discovered 14-3-3 zeta overexpression in many human primary cancers, such as breast, lung, and sarcoma, and in a majority of cancer cell lines. To determine 14-3-3 zeta involvement in breast cancer progression, we used immunohistochemical analysis to examine 14-3-3 zeta expression in human primary invasive breast carcinomas. High 14-3-3 zeta expression was significantly correlated with poor prognosis of breast cancer patients. Increased expression of 14-3-3 zeta was also significantly correlated with elevated PKB/Akt activation in patient samples. Thus, 14-3-3 zeta is a marker of poor prognosis in breast cancers. Furthermore, up-regulation of 14-3-3 zeta enhanced malignant transformation of cancer cells in vitro. ^ To determine the biological significance of 14-3-3 zeta in human cancers, small interfering RNAs (siRNA) were used to specifically block 14-3-3 zeta expression in cancer cells. 14-3-3 zeta siRNA inhibited cellular proliferation by inducing a G1 arrest associated with up-regulation of p27 KIP1 and p21CIP1 cyclin dependent kinase inhibitors. Reduced 14-3-3 zeta inhibited PKB/Akt activation while stimulating the p38 signaling pathway. Silencing 14-3-3 zeta expression also increased stress-induced apoptosis by caspase activation. Notably, 14-3-3 zeta siRNA inhibited transformation related properties of breast cancer cells in vitro and inhibited tumor progression of breast cancer cells in vivo. 14-3-3 zeta may be a key regulatory factor controlling multiple signaling pathways leading to tumor progression. ^ The data indicate 14-3-3 zeta is a major regulator of cell growth and apoptosis and may play a critical role in the development of multiple cancer types. Hence, blocking 14-3-3 zeta may be a promising therapeutic approach for numerous cancers. ^

The roles of costimulatory molecules in the cooperative effects of combination epinephrine and dexamethasone on type-1/type-2 cytokine production in tetanus-toxoid specific stimulated human peripheral blood mononuclear cells

A growing number of studies show strong associations between stress and altered immune function. In vivo studies of chronic and acute stress have demonstrated that cognitive stressors are strongly correlated with high circulating levels of catecholamines (CT) and corticosteroids (CS) that are associated with changes in type-1/type-2 cytokine expression. Although individual pharmacologic doses of CS and CT can inhibit the expression of T-helper 1 (Th1, type-1 like) and promote the production of T-helper 2 (Th2, type-2 like) cytokines in antigen-specific and mitogen stimulated human leukocyte cultures in vitro, little attention has been focused on the effects of combination physiologic-stress doses of CT and CS that may be more physiologically relevant. In addition, both in-vivo and in-vitro studies suggest that the differential expression of the B7 family of costimulatory molecules CD80 and CD86 may promote the expression of type-1 or type-2 cytokines, respectively. Furthermore, corticosteroids can influence the expression of β2-adrenergic receptors in various human tissues. We therefore investigated the combined effects of physiologic-stress doses of in vitro CT and CS upon the type-1/type-2 cytokine balance and expression of B7 costimulatory molecules of human peripheral blood mononuclear cells (PBMC) as a model to study the immunomodulatory effects of physiologic stress. Results demonstrated a significant decrease in type-1 cytokine expression and a significant increase in type-2 cytokine production in our CS+CT incubated cultures when compared to either CT or CS agents alone. In addition, we demonstrated the differential expression of CD80/CD86 in favor of CD86 at the cellular and population level as determined by flow cytometry in lipopolysaccharide stimulated human Monocytes. Furthermore, we developed flow cytometry based assays to detect total β2AR in human CD4+ T-lymphocytes that demonstrated decreased expression of β2AR in mitogen stimulated CD4+ T-lymphocytes in the presence of physiologic stress levels of CS and CT as single in vitro agents, however, when both CS and CT were combined, significantly higher expression of β2AR was observed. In summary, our in vitro data suggest that both CS and CT work cooperatively to shift immunity towards type-2 responses. ^

Reactive oxygen species-dependent mechanisms of N-(4-hydroxyphenyl)retinamide (4HPR)-induced apoptosis in human carcinoma cells

4HPR is a synthetic retinoid that has shown chemopreventive and therapeutic efficacy against premalignant and malignant lesions including oral leukoplakia, ovarian and breast cancer, and neuroblastoma. 4HPR induces apoptosis in various cancer cells and production of reactive oxygen species (ROS) has been suggested as a possible cause underlying these effects. However, the mechanisms governing these effects by 4HPR are not fully elucidated. In this study, we explored the mechanisms of 4HPR-induced ROS increase and apoptosis in human cancer cells. ^ First, we identified genes modulated by 4HPR using oligonucleotide gene expression arrays and found that they fall into specific functional canonical pathways and gene networks using Ingenuity Pathways Analysis®. Further analysis has shown that 4HPR induced up-regulation of Endoplasmic Reticulum (ER)-related genes such as Heat shock proteins 70 and 90 and the transcriptional factor, GADD153. These findings were validated using quantitative real-time PCR. ^ Second, we found that 4HPR induced extensive ER stress evidenced by dilation of the ER and endoribonuclease-mediated splicing and activation of the transcriptional factor, XBP-1. In addition, 4HPR induced the up-regulation of various ER stress-related genes and their protein products, as well as cleavage and activation of the ER specific Caspase-4. Concomitantly with XBP-1 splicing, all of these effects were dependent on ROS generation by 4HPR. Furthermore, chemical inhibition and RNA interference studies revealed a novel pro-apoptotic role for HSP70/A1A in 4HPR-mediated apoptosis. ^ Third, we observed rapid activation of the small GTPase Rac by 4HPR which was upstream of ROS generation. Inhibition of Rac activity or silencing of its expression by RNA interference inhibited ROS generation and apoptosis induction by 4HPR. siRNA targeting PAK1 and expression of a dominant negative Rac, decreased 4HPR-mediated ROS generation, while expression of a constitutive active Rac increased basal and 4HPR-induced ROS generation and PARP cleavage. Furthermore, metastatic cancer cells exhibited higher Rac activation, ROS generation, and cell growth inhibition due to 4HPR exposure compared to their primary cancer cell counterparts. ^ These findings provide novel insights into 4HPR-mediated ROS generation and apoptosis induction and support the use of ROS inducing agents such as 4HPR against metastatic cancer cells. ^

The effects of proteasome inhibition on angiogenesis and autophagy in human prostate cancer cells

The proteasome degrades approximately 80% of intracellular proteins to maintain homeostasis. Proteasome inhibition is a validated therapeutic strategy, and currently, proteasome inhibitor bortezomib is FDA approved for the treatment of MM and MCL. Specific pathways affected by proteasome inhibition have been identified, but mechanisms of the anti-tumor effects of proteasome inhibition are not fully characterized and cancer cells display marked heterogeneity in terms of their sensitivity to proteasome inhibitor induced cell death. ^ The antitumor effects of proteasome inhibition involve suppression of tumor angiogenesis and vascular endothelial growth factor (VEGF) expression, but the mechanisms involved have not been clarified. In this dissertation I investigated the mechanisms underlying the effects of two proteasome inhibitors, bortezomib and NPI-0052, on VEGF expression in human prostate cancer cells. I found that proteasome inhibitors selectively downregulated hypoxia inducible factor 1alpha (HIF-1α) protein and its transcriptional activity to inhibit VEGF expression. Mechanistic studies demonstrated that proteasome inhibitors mediate the induction of the unfolded protein response (UPR) and that downregulation of HIF-1α is caused by eukaryotic translation initiation factor 2α (eIF2α) phosphorylation and translation repression. Importantly, I showed that proteasome inhibitors activated the UPR in some cells but not in others. My observation may have implications for the design of combination regimens that are based on exploiting proteasome inhibitor-induced ER stress.^ Although proteasome inhibitors have shown modest activity on prostate cancer, there is general consensus that no single agent is likely to have significant activity in prostate cancer. In the second part of this dissertation I attempted to exploit the effects of proteasome inhibition on the UPR to design a combination therapy that would enhance cancer cell death. Autophagy is a lysosome dependent degradation pathway that functions to eliminate long-lived protein and subcellular structures. Targeting autophagy has been shown to inhibit tumors in preclinical studies. I found that inhibition of autophagy with chloroquine or 3-methyladenine enhanced proteasome inhibitor induced cell death and the effects were associated with increased intracellular stress as marked by aggresome formation. Multiple cancers appear to be resistant to proteasome inhibition treatment alone. The implications of synergy for the combined inhibition of autophagy and the proteasome would likely apply to other cancers aside from prostate cancer. ^

Evaluation of oxidative damage and renal distal tubule cell stress response following exposure to lindane

Lindane, or γ-hexachlorocyclohexane, is a chlorinated hydrocarbon pesticide that was banned from U.S. production in 1976, but until recently continued to be imported and applied for occupational and domestic purposes. Lindane is known to cause central nervous system (CNS), immune, cardiovascular, reproductive, liver, and kidney toxicity. The mechanism for which lindane interacts with the CNS has been elucidated, and involves antagonism of the γ-aminobutyric acid/benzodiazepine (GABAA/BZD) receptor. Antagonism of this receptor results in the inhibition of Cl- channel flux, with subsequent convulsions, seizures, and paralysis. This response makes lindane a desirable defense against arthropod pests in agriculture and the home. However, formulation and application of this compound can contribute to human toxicity. In conjunction with this exposure scenario, workers may be subject to both heat and physical stress that may increase their susceptibility to pesticide toxicity by altering their cellular stress response. The kidneys are responsible for maintaining osmotic homeostasis, and are exposed to agents that undergo urinary excretion. The mechanistic action of lindane on the kidneys is not well understood. Lindane, in other organ systems, has been shown to cause cellular damage by generation of free radicals and oxidative stress. Previous research in our laboratory has shown that lindane causes apoptosis in distal tubule cells, and delays renal stress response under hypertonic stress. Characterizing the mechanism of action of lindane under conditions of physiologic stress is necessary to understand the potential hazard cyclodiene pesticides and other organochlorine compounds pose to exposed individuals under baseline conditions, as well as under conditions of physiologic stress. We demonstrated that exposure to lindane results in oxidative damage and dysregulation of glutathione response in renal distal tubule (MDCK) cells. We showed that under conditions of hypertonic stress, lindane-induced oxidative stress resulted in early onset apoptosis and corresponding down-regulated expression of the anti-apoptotic protein, Bcl-xL. Thus, the interaction of lindane with renal peripheral benzodiazepine receptors (PBR) is associated with attenuation of cellular protective proteins, making the cell more susceptible to injury or death. ^

The influence of HIV/AIDS on the association between genital human papillomavirus and cervical cancer in HIV/AIDS infected women: A literature review

Background: With over 440 million cases of infections worldwide, genital HPV is the most frequent sexually transmitted infection. There are several types including high risk types 16, 18, 58 and 70 among others, which are known to cause cervical cell abnormality and if persistent, can lead to cervical cancer which globally, claims 288,000 lives annually. 33.4 million people worldwide are currently living with HIV/AIDS, with 22.4 million in sub-Saharan Africa where 70% of the female population living with HIV/AIDS is also found. Similar risk factors for HPV, cervical cancer and HIV/AIDS include early age at sexual debut, multiple sexual partners, infrequent condom use, history of STI and immune-suppression. ^ Objectives: To describe the role of HPV in cervical cancer development, to describe the influence of HIV/AIDS on HPV and in the development of cervical cancer and to describe the importance of preventive measures such as screening. ^ Methods: This is a literature review where data were analyzed qualitatively and a descriptive narrative style used to evaluate and present the information. The data came from searches using Pub Med, Cochrane Library, EBSCO Medline databases as well as websites such as the CDC and WHO. Articles selected were published in English over the last 10 years. Keywords used included: 'HPV, cervical cancer and HIV', 'HIV and HPV', 'HPV and cervical cancer', 'HPV infection', 'HPV vaccine', 'genital HPV', 'HIV and cervical cancer', 'prevalence of HIV and cervical cancer' and 'prevalence of cervical cancer'. ^ Results: Women with HIV/AIDS have multiple HPV types, persistent infection, are more likely to present with cervical neoplasia and are at higher risk for cervical cancer. Research also shows that HIV could affect the transmissibility of HPV and that HPV itself could also increase the susceptibility to HIV acquisition. ^ Conclusion: HIV, genital HPV and cervical cancer are all preventable. Need to emphasize programs that aim to increase HIV/AIDS, HPV and cervical cancer awareness. Stress importance of behavior modification such as frequent use of condoms, decreased sexual partners and delayed first intercourse. Facilitate programs for screening and treating HPV, male circumcision, effective management of HAART and HPV vaccination.^

AN INVESTIGATION OF THE IMPACT OF CHILDREN WITH BIRTH DEFECTS ON STRESS IN THEIR FAMILIES

A child with a birth defect places physical, financial and emotional stress upon the family. The purpose of this study was to assess the impact of a mildly handicapped child on the family's coping abilities.^ Two groups, 101 mothers of children with birth defects and 107 mothers of intact children, completed the Holroyd Questionnaire on Resources and Stress and the Luborsky Social Assets Scale. From these groups, 86 pairs were matched on four factors: the age (two to eight years) and sex of the study child and the mother's education and marital status.^ The children with birth defects had completed the diagnostic evaluation at the Meyer Center for Developmental Pediatrics, Texas Children's Hospital. Children with severe defects were excluded. The mean I.Q of the group was 88, s.d. 17; 17 children were mildly retarded and 35 had an I.Q. of 100 or above; areas of dysfunction included motor abnormalities, behavior disturbance, speech problems, and sensory impairments.^ The expected direction and statistically significant differences were obtained from the data for the matched pairs on the Q.R.S. scales. The mothers of children with a birth defect reported poor health, a negative attitude toward the child, being over-protective, financial problems and feeling a lack of social support and family integration. They perceived the child as socially obtrusive, limited as to occupational opportunities, and as having a difficult personality.^ The functioning levels of the handicapped children contributed to the respondent's problems. The child with behavior and speech problems but adequate intelligence was a situation which resulted in a poor health/mood of the mother. The mother's pessimism was related to the child's low intelligence.^ The social assets of the respondents with intact children were significantly higher than those of respondents of handicapped children. There was no relationship between the total social assets score and the scores on the Q.R.S. for mothers of handicapped children. These mothers did report poorer physical conditions, more smoking, and quarreling of their parents as they grew up. ^

Metabolic Regulation of mTOR Activation and Endoplasmic Reticulum Stress in the Heart

When subjected to increased workload, the heart responds metabolically by increasing its reliance on glucose and structurally by increasing the size of myocytes. Whether changes in metabolism regulate the structural remodeling process is unknown. A likely candidate for a link between metabolism and growth in the heart is the mammalian target of rapamycin (mTOR), which couples energy and nutrient metabolism to cell growth. Recently, sustained mTOR activation has also been implicated in the development of endoplasmic reticulum (ER) stress. We explored possible mechanisms by which acute metabolic changes in the hemodynamically stressed heart regulate mTOR activation, ER stress and cardiac function in the ex vivo isolated working rat heart. Doubling the heart’s workload acutely increased rates of glucose uptake beyond rates of glucose oxidation. The concomitant increase in glucose 6-phosphate (G6P) was associated with mTOR activation, endoplasmic reticulum (ER) stress and impaired contractile function. Both rapamycin and metformin restored glycolytic homeostasis, relieved ER stress and rescued contractile function. G6P and ER stress were also downregulated with mechanical unloading of failing human hearts. Taken together, the data support the hypothesis that metabolic remodeling precedes, triggers, and sustains structural remodeling of the heart and implicate a critical role for G6P in load-induced contractile dysfunction, mTOR activation and ER stress. In general terms, the intermediary metabolism of energy providing substrates provides signals for the onset and progression of hypertrophy and heart failure.

Chronic stress promotes tumor growth through increased BDNF production and neo-innervation

Background: Activation of the sympathetic nervous system (SNS) in response to chronic biobehavioral stress results in high levels of catecholamines and persistent activation of adrenergic signaling, which promotes tumor growth and progression. However it is unknown how catecholamine levels within the tumor exceed systemic levels in circulation. I hypothesized that neo-innervation of tumors is required for stress-mediated effects on tumor growth. Results: First, I examined whether sympathetic nerves are present in human ovarian cancer samples as well as orthotopic ovarian cancer models. Immunohistochemical (IHC) staining for neurofilament revealed that catecholaminergic neurons are present within tumor tissue. In order to determine whether chronic stress affects the density of nerves in the tumor, I utilized an orthotopic mouse model of ovarian cancer that was exposed to daily restraint stress. IHC analysis revealed that nerve density in tumors increased by more than three-fold in stressed animals versus non-stressed controls. IHC analysis suggested that this results from both recruitment of existing neurons (axonogenesis) as well as new neuron formation (neurogenesis) within the tumor. To determine how tumors are recruiting nerve growth, I utilized a PCR array analysis of 84 nerve growth related genes and their receptors, which showed that stimulation of the SKOV3 ovarian cancer cell line with norepinephrine (NE) leads to increased expression of several neurotrophins, including brain-derived neurotrophic factor (BDNF). Neurite extension assays showed that media conditioned by ovarian cancer cell lines is capable of inducing neurite outgrowth in differentiated neuron-like PC12 cells, and NE treatment of cancer cells potentiates this effect. Norepinephrine-induced neurite extension was abolished after BDNF silencing by siRNA, suggesting that BDNF is critical to tumor cell-induced nerve growth. in vivo BDNF inhibition resulted in complete abrogation of stress-induced increases in tumor weight and nerve density, as well as downstream markers of stress. Discussion: These studies indicate that adrenergic signalling induced by chronic stress promotes neo-innervation in the tumor microenvironment. This results in a mutually beneficial relationship between the tumor cells and neurons. This work is crucial for providing a link between chronic stress and its effects on the tumor and its microenvironment. The data shown here aims to open new venues that can be used in development of therapies designed to block the stress effects on tumor growth.