Depressed type 1 cytokine synthesis by superantigen-activated CD4+ T cells of women with human papillomavirus-related high-grade squamous intraepithelial lesions.

Carcinoma of the cervix is causally related to infection with the human papillomavirus (HPV), and T cells play a pivotal role in the immune response of the host to rid itself of HPV infection. Therefore, we assessed the T-cell function of women with HPV-related cervical neoplasia against a superantigen, Staphylococcus enterotoxin B (SEB). Each woman provided a cervical brush specimen for HPV DNA testing and Papanicolaou (Pap) smears for the staging of cervical lesions. They also provided a blood specimen for determination of the ability of CD4(+) T and CD8(+) T cells to synthesize Th1 (interleukin-2 [IL-2], gamma interferon [IFN-gamma], and tumor necrosis factor alpha [TNF-alpha]) and Th2 (IL-10) cytokines in response to activation with SEB. Compared with control subjects with self-attested negative Pap smears, women with high-grade squamous intraepithelial lesions (HSIL) had significantly lower percentages of activated CD4(+) T cells that produced IL-2 (P = 0.045), IFN-gamma (P = 0.040), and TNF-alpha (P = 0.015) and a significantly lower percentage of activated CD8(+) T cells that produced IL-2 (P < 0.01). These data indicate that women with HPV-related cervical HSIL show a decrease in Th1 cytokine production by activated CD4(+) T cells and suggested that compromised T-helper functions may negatively impact the function of cytotoxic CD8(+) T cells.

Morphoproteomic analysis reveals an overexpressed and constitutively activated phospholipase D1-mTORC2 pathway in endometrial carcinoma.

The mammalian target of rapamycin (MTOR) assembles into two distinct complexes: mTOR complex 1 (mTORC1) is predominantly cytoplasmic and highly responsive to rapamycin, whereas mTOR complex 2 (mTORC2) is both cytoplasmic and nuclear, and relatively resistant to rapamycin. mTORC1 and mTORC2 phosphorylatively regulate their respective downstream effectors p70S6K/4EBP1, and Akt. The resulting activated mTOR pathways stimulate protein synthesis, cellular proliferation, and cell survival. Moreover, phospholipase D (PLD) and its product, phosphatidic acid (PA) have been implicated as one of the upstream activators of mTOR signaling. In this study, we investigated the activation status as well as the subcellular distribution of mTOR, and its upstream regulators and downstream effectors in endometrial carcinomas (ECa) and non-neoplastic endometrial control tissue. Our data show that the mTORC2 activity is selectively elevated in endometrial cancers as evidenced by a predominant nuclear localization of the activated form of mTOR (p-mTOR at Ser2448) in malignant epithelium, accompanied by overexpression of nuclear p-Akt (Ser473), as well as overexpression of vascular endothelial growth factor (VEGF)-A isoform, the latter a resultant of target gene activation by mTORC2 signaling via hypoxia-inducible factor (HIF)-2alpha. In addition, expression of PLD1, one of the two major isoforms of PLD in human, is increased in tumor epithelium. In summary, we demonstrate that the PLD1/PA-mTORC2 signal pathway is overactivated in endometrial carcinomas. This suggests that the rapamycin-insensitive mTORC2 pathway plays a major role in endometrial tumorigenesis and that therapies designed to target the phospholipase D pathway and components of the mTORC2 pathway should be efficacious against ECa.

Morphoproteomic evidence of constitutively activated and overexpressed mTOR pathway in cervical squamous carcinoma and high grade squamous intraepithelial lesions.

Human papilloma virus (HPV) infection of the uterine cervix is linked to the pathogenesis of cervical cancer. Preclinical in vitro and in vivo studies using HPV-containing human cervical carcinoma cell lines have shown that the mammalian target of rapamycin (mTOR) inhibitor, rapamycin, and epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor, erlotinib, can induce growth delay of xenografts. Activation of Akt and mTOR are also observed in cervical squamous cell carcinoma and, the expression of phosphorylated mTOR was reported to serve as a marker to predict response to chemotherapy and survival of cervical cancer patients. Therefore, we investigated: a) the expression level of EGFR in cervical squamous cell carcinoma (SCC) and high-grade squamous intraepithelial lesions (HSIL) versus non-neoplastic cervical squamous epithelium; b) the state of activation of the mTOR pathway in these same tissues; and c) any impact of these signal transduction molecules on cell cycle. Formalin-fixed paraffin-embedded tissue microarray blocks containing 20 samples each of normal cervix, HSIL and invasive SCC, derived from a total of 60 cases of cervical biopsies and cervical conizations were examined. Immunohistochemistry was utilized to detect the following antigens: EGFR; mTOR pathway markers, phosphorylated (p)-mTOR (Ser2448) and p-p70S6K (Thr389); and cell cycle associated proteins, Ki-67 and S phase kinase-associated protein (Skp)2. Protein compartmentalization and expression were quantified in regard to proportion (0-100%) and intensity (0-3+). Mitotic index (MI) was also assessed. An expression index (EI) for pmTOR, p-p70S6K and EGFR, respectively was calculated by taking the product of intensity score and proportion of positively staining cells. We found that plasmalemmal EGFR expression was limited to the basal/parabasal cells (2-3+, EI = 67) in normal cervical epithelium (NL), but was diffusely positive in all HSIL (EI = 237) and SCC (EI 226). The pattern of cytoplasmic p-mTOR and nuclear p-p70S6K expression was similar to that of EGFR; all showed a significantly increased EI in HSIL/SCC versus NL (p<0.02). Nuclear translocation of p-mTOR was observed in all SCC lesions (EI = 202) and was significantly increased versus both HSIL (EI = 89) and NL (EI = 54) with p<0.015 and p<0.0001, respectively. Concomitant increases in MI and proportion of nuclear Ki-67 and Skp2 expression were noted in HSIL and SCC. In conclusion, morphoproteomic analysis reveals constitutive activation and overexpression of the mTOR pathway in HSIL and SCC as evidenced by: increased nuclear translocation of pmTOR and p-p70S6K, phosphorylated at putative sites of activation, Ser2448 and Thr389, respectively; correlative overexpression of the upstream signal transducer, EGFR, and increases in cell cycle correlates, Skp2 and mitotic indices. These results suggest that the mTOR pathway plays a key role in cervical carcinogenesis and targeted therapies may be developed for SCC as well as its precursor lesion, HSIL.

The role of peroxisome proliferator-activated receptors in the development and physiology of gametes and preimplantation embryos.

In several species, a family of nuclear receptors, the peroxisome proliferator-activated receptors (PPARs) composed of three isotypes, is expressed in somatic cells and germ cells of the ovary as well as the testis. Invalidation of these receptors in mice or stimulation of these receptors in vivo or in vitro showed that each receptor has physiological roles in the gamete maturation or the embryo development. In addition, synthetic PPAR gamma ligands are recently used to induce ovulation in women with polycystic ovary disease. These results reveal the positive actions of PPAR in reproduction. On the other hand, xenobiotics molecules (in herbicides, plasticizers, or components of personal care products), capable of activating PPAR, may disrupt normal PPAR functions in the ovary or the testis and have consequences on the quality of the gametes and the embryos. Despite the recent data obtained on the biological actions of PPARs in reproduction, relatively little is known about PPARs in gametes and embryos. This review summarizes the current knowledge on the expression and the function of PPARs as well as their partners, retinoid X receptors (RXRs), in germ cells and preimplantation embryos. The effects of natural and synthetic PPAR ligands will also be discussed from the perspectives of reproductive toxicology and assisted reproductive technology.

Tumor cell-associated epitopes recognized by lymphokine-activated killers (LAK)

Interleukin-2 activated lymphocytes, designated lymphokine-activated killers (LAK), acquire the unique capacity to express potent cytologic activity against a broad spectrum of abnormal and/or transformed NK-sensitive and NK-resistant target cells while sparing normal cell types. Investigations into the target spectra exhibited by cloned effector cells indicate that LAK cells express a polyspecific recognition mechanism that identifies an undefined class of cell surface-associated molecules expressed on susceptible targets. This report extends our previous investigations into the biochemical nature of these molecules by characterizing the functional role of two tumor cell-surface-associated epitopes implicated in conferring target cells with susceptibility to LAK-mediated cytotoxicity. The first moiety is implicated in the formation of effector/target cell conjugates. This binding ligand is preferentially expressed on tumor cells relative to LAK-resistant PBL target cells, sensitive to trypsin treatment, resistant to functional inactivation by heat- and detergent-induced conformational changes, and does not require N-linked glycosylation to maintain binding activity. In contrast, a carbohydrate-associated epitope represents the second tumor-associated molecule required for target cell susceptibility to LAK cells. Specifically, N-linked glyoprotein synthesis represents an absolute requirement for post-trypsin recovery of target cell susceptibility. The minimal N-linked oligosaccharide residue capable of restoring this second signal has been identified as a high mannose structure. Although ultimately required for tumor cell susceptibility, as measured in $\sp{51}$Cr-release assays, this N-glycan-associated molecule is not required for the differential tumor cell binding expressed by LAK cells. Furthermore, N-glycan expression is not adequate in itself to confer target cell susceptibility. Additional categories of cell surface components have been investigated, including O-linked oligosaccharides, and glycosaminoglycans, without identifying additional moieties relevant to target cell recognition. Collectively, these data suggest that tumor cell recognition by LAK cells is dependent on cell surface presentation of two epitopes: a trypsin-sensitive molecule that participates in the initial conjugate formation and an N-glycan-associated moiety that is involved in a post-binding event required for target cell killing. ^

INHIBITION OF THE MITOCHONDRIAL RESPIRATION OF TUMOR CELLS BY SOLUBLE FACTORS RELEASED BY ACTIVATED MACROPHAGES (CYTOTOXICITY, ELECTRON TRANSPORT, MONOCYTE)

Particular interest has been directed towards the macrophage as a primary antineoplastic cell due to its tumoricidal properties in vitro and the observation that an inverse relationship exists between the number of macrophages infiltrating a tumor and metastatic potential. The mechanism of macrophage-mediated injury of tumor cells remains unknown. Recently, it has been shown that injured tumor cells have defective mitochondrial respiration. Our studies have shown that activated macrophages can release soluble factors which can alter tumor cell respiration.^ The effects of a conditioned supernatant (CS) from cultures of activated macrophages on tumor cell (TC) mitochondrial respiration was studied. CS was obtained by incubation of BCG-elicited, murine peritoneal macrophage with RPMI-1640 supplemented with 10% FCS and 50 ng/ml bacterial endotoxin. This CS was used to treat cultures of EMT-6 TC for 24 hours. Mitochondrial respiration was measured polarigraphically using a Clark-type oxygen electrode. Cell growth rate was assessed by ('3)H-Thymidine incorporation. Exposure of EMT-6 TC to CS resulted in the inhibition of malate and succinate oxidation 76.6% and 72.9%, respectively. While cytochrome oxidase activity was decreased 61.1%. This inhibition was accompanied by a 98.8% inhibition of DNA synthesis (('3)H-Thymidine incorporation). Inhibition was dose-related with a 21.3% inhibition of succinate oxidase from a 0.3 ml dose of CS and a 50% inhibition with 1.0 mls. Chromatography of CS on Sephacryl S-200 resulted in isolation of an 80,000 and a 55,000 dalton component which contained the respiration inhibiting activity (RIF). These factors were distinct from a 120,000 dalton cytolytic factor determined by bioassay on Actinomycin-D treated L929 cells. RIF activity was also distinct from several other cytostatic factors but was itself associated with 2 peaks of cytostatic activity. Characterization of the RIF activity showed that it was destroyed by trypsin and heat (100(DEGREES)C, 5 min). It was stable over a broad range of pH (4-9) and its production was inhibited by cycloheximide. The RIF did not have a direct effect on isolated mitochondria of TC nor did it induce the formation of a stable intracellular toxin for mitochondria.^ In conclusion, activated macrophages synthesize and secrete an 80,000 and a 55,000 dalton protein which inhibits the mitochondrial metabolism of TC. These factors induce a cytostatic but not a cytolytic effect on TC.^ The macrophage plays a role in the control of normal and tumor cell growth and in tissue involution. Inhibition of respiration may be one mechanism used by macrophages to control cell growth.^

p21 activated kinase 5 links multiple GTPase signaling pathways at mitochondria

The p21-activated kinase 5 (PAK5) is a serine/threonine protein kinase associated with the group 2 subfamily of PAKs. Although our understanding about PAK5 is very limited, it is receiving increasing interest due to its tissue specific expression pattern and important signaling properties. PAK5 is highly expressed in brain. Its overexpression induces neurite outgrowth in neuroblastoma cells and promotes survival in fibroblasts. ^ The serine/threonine protein kinase Raf-1 is an essential mediator of Ras-dependent signaling that controls the ERK/MAPK pathway. In contrast to PAK5, Raf-1 has been the subject of intensive investigation. However due to the complexity of its activation mechanism, the biological inputs controlling Raf-1 activation are not fully understood. ^ PAKs 1-3 are the known kinases responsible for phosphorylation of Raf-1 on serine 338, which is a crucial phosphorylation site for Raf-1 activation. However, dominant negative versions of these kinases do not block EGF-induced Raf-1 activation, indicating that other kinases may regulate the phosphorylation of Raf-1 on serine 338. ^ This thesis work was initiated to test whether the group 2 PAKs 4, 5 and 6 are responsible for EGF-induced Raf-1 activation. We found that PAK5, and to a lesser extent PAK4, can activate Raf-1 in cells. Our studies thereafter focused on PAK5. With the progress of our study we found that PAK5 does not significantly stimulate serine 338 phosphorylation of Triton X-100 soluble Raf-1. PAK5, however, constitutively and specifically associates with Raf-1 and targets it to a Triton X-100 insoluble, mitochondrial compartment, where PAK5 phosphorylates serine 338 of Raf-1. We further demonstrated that endogenous PAK5 and Raf-1 colocalize in Hela cells at the mitochondrial outer membrane. In addition, we found that the mitochondria-targeting of PAK5 is determined by its C-terminal kinase domain plus the upstream proximal region, and facilitated by the N-terminal p21 binding domain. We also demonstrated that Rho GTPases Cdc42 and RhoD associate with and regulate the subcellular localization of PAK5. Taken together, this work suggests that the mitochondria-targeting of PAK5 may link Ras and Rho GTPase-mediated signaling pathways, and sheds light on aspects of PAK5 signaling that may be important for regulating neuronal homeostasis. ^

Protease-activated receptor-1 signaling plays a major role in melanoma growth and metastasis

Overexpression of the thrombin receptor (Protease-Activated-Receptor-1), PAR-1, in cell lines and tissue specimens correlates with the metastatic potential of human melanoma. Utilizing lentiviral shRNA to stably silence PAR-1 in metastatic melanoma cell lines results in decreased tumor growth and lung metastasis in vivo. Since the use of viral technology is not ideal for clinical therapies, neutral liposomes (DOPC) were utilized as a delivery vehicle for PAR-1 siRNA. Our data suggest that PAR-1 siRNA-DOPC treatment by systemic delivery significantly decreases tumor growth and lung metastasis in nude mice. Concomitant decreases in angiogenic and invasive factors (IL-8, VEGF, MMP-2) were observed in PAR-1 siRNA-DOPC-treated mice. Utilizing a cDNA microarray platform, several novel PAR-1 downstream target genes were identified, including Connexin 43 (Cx-43) and Maspin. Cx-43, known to be involved in tumor cell diapedesis and attachment to endothelial cells, is decreased after PAR-1 silencing. Furthermore, the Cx-43 promoter activity was significantly inhibited in PAR-1-silenced cells suggesting transcriptional regulation of Cx-43 by PAR-1. ChIP analysis revealed a reduction in SP-1 and AP-1 binding to the Cx-43 promoter. Moreover, melanoma cell attachment to HUVEC was significantly decreased in PAR-1-silenced cells as well as in Cx-43 shRNA transduced cells. As both SP-1 and AP-1 transcription factors act as positive regulators of Cx-43, our data provide a novel mechanism for the regulation of Cx-43 expression by PAR-1. Maspin, a serine protease inhibitor with tumor-suppressor function, was found to be upregulated after PAR-1 silencing. Our results indicate that PAR-1 transcriptionally regulates Maspin, as the promoter activity was significantly increased after PAR-1 silencing. ChIP analysis revealed that silencing PAR-1 increased binding of Ets and c-Jun to the Maspin promoter. As Maspin was recently found to be a tumor-suppressor in melanoma by reducing the invasive capacity of melanoma cells, invasion assays revealed a decrease in invasion after PAR-1 silencing and in cells transduced with a Maspin expression vector. We propose that PAR-1 is key to the progression and metastasis of melanoma in part by regulating the expression of Cx-43 and Maspin. Taken together, we propose that PAR-1 is an attractive target for the treatment of melanoma.^

Regulation of Protein Degradation in the Heart by AMP-Activated Protein Kinase

The degradation of proteins by the ubiquitin proteasome system is essential for cellular homeostasis in the heart. An important regulator of metabolic homeostasis is AMP-activated protein kinase (AMPK). During nutrient deprivation, AMPK is activated and intracellular proteolysis is enhanced through the ubiquitin proteasome system (UPS). Whether AMPK plays a role in protein degradation through the UPS in the heart is not known. Here I present data in support of the hypothesis that AMPK transcriptionally regulates key players in the UPS, which, under extreme conditions can be detrimental to the heart. The ubiquitin ligases MAFbx /Atrogin-1 and MuRF1, key regulators of protein degradation, and AMPK activity are increased during nutrient deprivation. Pharmacologic and genetic activation of AMPK is sufficient for the induction of MAFbx/Atrogin-1 and MuRF1 in cardiomyocytes and in the heart in vivo. Comprehensive experiments demonstrate that the molecular mechanism by which AMPK regulates MuRF1 expression is through the transcription factor myocyte enhancer factor 2 (MEF2), which is involved in stress response and cardiomyocyte remodeling. MuRF1 is required for AMPK-mediated protein degradation through the UPS in cardiomyocytes. Consequently, the absence of MuRF1 during chronic fasting preserves cardiac function, possibly by limiting degradation of critical metabolic enzymes. Furthermore, during cardiac hypertrophy, chronic activation of AMPK also leads to cardiac dysfunction, possibly through enhanced protein degradation and metabolic dysregulation. Collectively, my findings demonstrate that AMPK regulates expression of ubiquitin ligases which are required for UPS-mediated protein degradation in the heart. Based on these results, I propose that specific metabolic signals may serve as modulators of intracellular protein degradation in the heart.