Growth and differentiation of bone marrow hematopoietic cells in mice bearing Ehrlich ascite tumor and treated with Dicyclopentadienildichiorotitanium (IV)

In this work we have:investigated the growth and differentiation of bone marrow stem cells in mice bearing Ehrlich ascites tumor-and treated with three dose-regimens of Dicyclopentadienyldichlorotitanium (IV) (DDCT). We also: studied the presence of colony stimulating factors In the serum of PDCT-treated animals as well-as the effects-of the drug on the survival of the tumor-bearing mice. The-results demonstrated that the myelosuppression developed in the tumor-bearing animals is prevented by the administration:of 1, 2 or 3 doses of 15 mg/kg DDCT. In the treatment with three doses, however, 23 % of the animals died. Moreover, DDCT treatment in normal animals resulted in increased numbers of CFU-GM. We observed the presence of stimulating factors in the serum of drug-treated animals which induced the growth and differentiation of bone marrow progenitor cells from normal animals in vitro. on the other hand, in vitro addition of the drug to these cultures had no effect. Thus, we conclude that the drug protects against the myelosuppression induced by the tumor and that this protection may be related to an indirect action of the drug. (C) 1998 International Society for Immunopharmacology. Published by Elsevier B.V. Ltd.

Evaluation of prognostic factors and survival rates in malignant feline mammary gland neoplasms

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of addition of a bird repellent to fish diets on their growth and bioaccumulation

The effects of adding the nonlethal bird repellent methyl anthranilate (MA), at levels of 100 and 1000 mg/kg, to fish feed on the bioaccumulation and growth of juvenile (10 g) hybrid striped bass (Morone chrysops x M. saxatilis) and juvenile (1g) African cichlid fish Aulonocara jacobfreibergi were investigated under laboratory conditions. The bird repellent did not have any effect on the fish growth or survival over a period of 6 weeks. MA residues at low levels of 11.2 ± 2.6 mg/g were found in lipophilic tissues (liver) of MA-fed fish. Control fish, which had no MA added to their diet, had a much lower level of 0.6 ± 0.3 mg/g MA in their liver. Fish muscle was found to contain negligible MA residues, while the outer body surface mucus did not contain any MA. Following a 6-week depuration period, during which the previously MA-fed hybrid striped bass were fed a feed to which no MA was added, the levels of MA residues detected were reduced by one order of magnitude.

Platelet-activating factor receptor (PAF-R)-dependent pathways control tumour growth and tumour response to chemotherapy

Abstract Background Phagocytosis of apoptotic cells by macrophages induces a suppressor phenotype. Previous data from our group suggested that this occurs via Platelet-activating factor receptor (PAF-R)-mediated pathways. In the present study, we investigated the impact of apoptotic cell inoculation or induction by a chemotherapeutic agent (dacarbazine, DTIC) on tumour growth, microenvironmental parameters and survival, and the effect of treatment with a PAF-R antagonist (WEB2170). These studies were performed in murine tumours: Ehrlich Ascitis Tumour (EAT) and B16F10 melanoma. Methods Tumour growth was assessed by direct counting of EAT cells in the ascitis or by measuring the volume of the solid tumour. Parameters of the tumour microenvironment, such as the frequency of cells expressing cyclo-oxygenase-2 (COX-2), caspase-3 and galectin-3, and microvascular density, were determined by immunohistochemistry. Levels of vascular endothelium growth factor (VEGF) and prostaglandin E2 (PGE2) were determined by ELISA, and levels of nitric oxide (NO) by Griess reaction. PAF-R expression was analysed by immunohistochemistry and flow cytometry. Results Inoculation of apoptotic cells before EAT implantation stimulated tumour growth. This effect was reversed by in vivo pre-treatment with WEB2170. This treatment also reduced tumour growth and modified the microenvironment by reducing PGE2, VEGF and NO production. In B16F10 melanoma, WEB2170 alone or in association with DTIC significantly reduced tumour volume. Survival of the tumour-bearing mice was not affected by WEB2170 treatment but was significantly improved by the combination of DTIC with WEB2170. Tumour microenvironment elements were among the targets of the combination therapy since the relative frequency of COX-2 and galectin-3 positive cells and the microvascular density within the tumour mass were significantly reduced by treatment with WEB2170 or DTIC alone or in combination. Antibodies to PAF-R stained the cells from inside the tumour, but not the tumour cells grown in vitro. At the tissue level, a few cells (probably macrophages) stained positively with antibodies to PAF-R. Conclusions We suggest that PAF-R-dependent pathways are activated during experimental tumour growth, modifying the microenvironment and the phenotype of the tumour macrophages in such a way as to favour tumour growth. Combination therapy with a PAF-R antagonist and a chemotherapeutic drug may represent a new and promising strategy for the treatment of some tumours.

Early life of the abalone Haliotis tuberculata coccínea : development, settlement and growth

Programa de doctorado: Acuicultura

Impact of CD39 and purinergic signalling on the growth and metastasis of colorectal cancer

Despite improvements in prevention and management of colorectal cancer (CRC), uncontrolled tumor growth with metastatic spread to distant organs remains an important clinical concern. Genetic deletion of CD39, the dominant vascular and immune cell ectonucleotidase, has been shown to delay tumor growth and blunt angiogenesis in mouse models of melanoma, lung and colonic malignancy. Here, we tested the influence of CD39 on CRC tumor progression and metastasis by investigating orthotopic transplanted and metastatic cancer models in wild-type BALB/c, human CD39 transgenic and CD39 deficient mice. We also investigated CD39 and P2 receptor expression patterns in human CRC biopsies. Murine CD39 was expressed by endothelium, stromal and mononuclear cells infiltrating the experimental MC-26 tumors. In the primary CRC model, volumes of tumors in the subserosa of the colon and/or rectum did not differ amongst the treatment groups at day 10, albeit these tumors rarely metastasized to the liver. In the dissemination model, MC-26 cell line-derived hepatic metastases grew significantly faster in CD39 over-expressing transgenics, when compared to CD39 deficient mice. Murine P2Y2 was significantly elevated at both mRNA and protein levels, within the larger liver metastases obtained from CD39 transgenic mice where changes in P2X7 levels were also noted. In clinical samples, lower levels of CD39 mRNA in malignant CRC tissues appeared associated with longer duration of survival and could be linked to less invasive tumors. The modulatory effects of CD39 on tumor dissemination and differential levels of CD39, P2Y2 and P2X7 expression in tumors suggest involvement of purinergic signalling in these processes. Our studies also suggest potential roles for purinergic-based therapies in clinical CRC.

Telomeres: Linking stress and survival, ecology and evolution.

Telomeres are protective structures at the ends of eukaryotic chromosomes. The loss of telomeres through cell division and oxidative stress is related to cellular aging, organismal growth and disease. In this way, telomeres link molecular and cellular mechanisms with organismal processes, and may explain variation in a number of important life-history traits. Here, we discuss how telomere biology relates to the study of physiological ecology and life history evolution. We emphasize current knowledge on how telomeres may relate to growth, survival and lifespan in natural populations. We finish by examining interesting new connections between telomeres and the glucocorticoid stress response. Glucocorticoids are often employed as indices of physiological condition, and there is evidence that the glucocorticoid stress response is adaptive. We suggest that one way that glucocorticoids impact organismal survival is through elevated oxidative stress and telomere loss. Future work needs to establish and explore the link between the glucocorticoid stress response and telomere shortening in natural populations. If a link is found, it provides an explanatory mechanism by which environmental perturbation impacts life history trajectories.

Novel agents targeting the IGF-1R/PI3K pathway impair cell proliferation and survival in subsets of medulloblastoma and neuroblastoma

The receptor tyrosine kinase (RTK)/phosphoinositide 3-kinase (PI3K) pathway is fundamental for cancer cell proliferation and is known to be frequently altered and activated in neoplasia, including embryonal tumors. Based on the high frequency of alterations, targeting components of the PI3K signaling pathway is considered to be a promising therapeutic approach for cancer treatment. Here, we have investigated the potential of targeting the axis of the insulin-like growth factor-1 receptor (IGF-1R) and PI3K signaling in two common cancers of childhood: neuroblastoma, the most common extracranial tumor in children and medulloblastoma, the most frequent malignant childhood brain tumor. By treating neuroblastoma and medulloblastoma cells with R1507, a specific humanized monoclonal antibody against the IGF-1R, we could observe cell line-specific responses and in some cases a strong decrease in cell proliferation. In contrast, targeting the PI3K p110α with the specific inhibitor PIK75 resulted in broad anti-proliferative effects in a panel of neuro- and medulloblastoma cell lines. Additionally, sensitization to commonly used chemotherapeutic agents occurred in neuroblastoma cells upon treatment with R1507 or PIK75. Furthermore, by studying the expression and phosphorylation state of IGF-1R/PI3K downstream signaling targets we found down-regulated signaling pathway activation. In addition, apoptosis occurred in embryonal tumor cells after treatment with PIK75 or R1507. Together, our studies demonstrate the potential of targeting the IGF-1R/PI3K signaling axis in embryonal tumors. Hopefully, this knowledge will contribute to the development of urgently required new targeted therapies for embryonal tumors.

Outside-in signaling through integrins and cadherins: a central mechanism to control epidermal growth and differentiation?

The process of epidermal renewal persists throughout the entire life of an organism. It begins when a keratinocyte progenitor leaves the stem cell compartment, undergoes a limited number of mitotic divisions, exits the cell cycle, and commits to terminal differentiation. At the end of this phase, the postmitotic keratinocytes detach from the basement membrane to build up the overlaying stratified epithelium. Although highly coordinated, this sequence of events is endowed with a remarkable versatility, which enables the quiescent keratinocyte to reintegrate into the cell cycle and become migratory when necessary, for example after wounding. It is this versatility that represents the Achilles heel of epithelial cells allowing for the development of severe pathologies. Over the past decade, compelling evidence has been provided that epithelial cancer cells achieve uncontrolled proliferation following hijacking of a "survival program" with PI3K/Akt and a "proliferation program" with growth factor receptor signaling at its core. Recent insights into adhesion receptor signaling now propose that integrins, but also cadherins, can centrally control these programs. It is suggested that the two types of adhesion receptors act as sensors to transmit extracellular stimuli in an outside-in mode, to inversely modulate epidermal growth factor receptor signaling and ensure cell survival. Hence, cell-matrix and cell-cell adhesion receptors likely play a more powerful and wide-ranging role than initially anticipated. This Perspective article discusses the relevance of this emerging field for epidermal growth and differentiation, which can be of importance for severe pathologies such as tumorigenesis and invasive metastasis, as well as psoriasis and Pemphigus vulgaris.

Synthesis and cellular characterization of novel isoxazolo- and thiazolohydrazinylidene-chroman-2,4-diones on cancer and non-cancer cell growth and death

Coumarins are extensively studied anticoagulants that exert additional effects such as anticancerogenic and even anti-inflammatory. In order to find new drugs with anticancer activities, we report here the synthesis and the structural analysis of new coumarin derivatives which combine the coumarin core and five member heterocycles in hydrazinylidene-chroman-2,4-diones. The derivatives were prepared by derivatization of the appropriate heterocyclic amines which were used as electrophiles to attack the coumarin ring. The structures were characterized by spectroscopic techniques including IR, NMR, 2D-NMR and MS. These derivatives were further characterized especially in terms of a potential cytotoxic and apoptogenic effect in several cancer cell lines including the breast and prostate cancer cell lines MCF-7, MDA-MB-231, PC-3, LNCaP, and the monocytic leukemia cell line U937. Cell viability was determined after 48 h and 72 h of treatment with the novel compounds by MTT assay and the 50% inhibitory concentrations (EC50 values) were determined. Out of the 8 novel compounds screened for reduced cell viability, 4c, 4d and 4e were found to be the most promising and effective ones having EC50 values that were several fold reduced when compared to the reference substance 4-hydroxycoumarin. However, the effects were cancer cell line dependent. The breast cancer MDA-MB-231 cells, the prostate cancer LNCaP cells, and U937 cells were most sensitive, MCF-7 cells were less sensitive, and PC-3 cells were more resistant. Reduced cell viability was accompanied by increased apoptosis as shown by PARP-1 cleavage and reduced activity of the survival protein kinase Akt. In summary, this study has identified three novel coumarin derivatives that in comparison to 4-hydroxycoumarin have a higher efficiency to reduce cancer cell viability and trigger apoptosis and therefore may represent interesting novel drug candidates

Theileria parva: taking control of host cell proliferation and survival mechanisms.

The intracellular parasite Theileria parva infects and transforms bovine T-cells, inducing their uncontrolled proliferation and spread in non-lymphoid as well as lymphoid tissues. This parasite-induced transformation is the predominant factor contributing to the pathogenesis of a lymphoproliferative disease, called East Coast fever. T. parva-transformed cells become independent of antigenic stimulation or exogenous growth factors. A dissection of the signalling pathways that are activated in T. parva-infected cells shows that the parasite bypasses signalling pathways that normally emanate from the T-cell antigen receptor to induce continuous proliferation. This review concentrates on the influence of the parasite on the state of activation of the mitogen-activated protein kinase (MAPK), NF-kappaB and phosphoinositide-3-kinase (PI3-K) pathways in the host cell. Of the MAPKs, JNK, but not ERK or p38, is active, inducing constitutive activation of the transcription factors AP-1 and ATF-2. A crucial step in the transformation process is the persistent activation of the transcription factor NF-kappaB, which protects T. parva-transformed cells from spontaneous apoptosis accompanying the transformation process. Inhibitor studies also suggest an important role for the lipid kinase, PI-3K, in the continuous proliferation of T. parva-transformed lymphocytes.

Growth regulatory role of R -PTPmu in normal and neoplastic epithelial cells

Cellular oncogenes and tumor suppressor genes regulate cellular adhesion and proliferation, two important events in malignant transformation. Even though receptor-like protein tyrosine phosphatases (R-PTPs) can influence these events, their role in malignant transformation has not been studied. The major goal of this study was to determine whether downregulation of R-PTP$\mu$ expression in lung epithelial cells is associated with or causal to neoplastic transformation. Examination of R-PTP$\mu$ expression in normal and carcinoma cells demonstrated that lung epithelial cells expressed R-PTP$\mu$ whereas lung carcinoma cells did not, and that incubation with TGF-$\alpha$ and HGF induced a two fold increase in R-PTP$\mu$ mRNA expression. To associate the expression of R-PTP$\mu$ with neoplastic transformation, we transfected lung epithelial cells with the H-ras oncogene. Transformation resulted in the activation of the MAPK signal transduction pathway, the hyperphosphorylation of c-met, and the production of HGF. Upon analysis of R-PTP$\mu$ expression, we observed a significant decrease in R-PTP$\mu$ mRNA and protein levels suggesting that transformation can directly or indirectly downregulate the expression of R-PTP$\mu.$ TGF-$\beta$ reversed the H-ras transformed phenotype, an event directly correlated with upregulation of R-PTP$\mu.$ To provide a casual relationship between R-PTP$\mu$ and cessation of tumor cell growth, we transfected carcinoma cells with the wild type R-PTP$\mu$ cDNA. Transiently expressing cells were selected by FACS using the mAb 3D7 and plated into individual wells. Carcinoma cells positive for R-PTP$\mu$ expression did not grow into colonies whereas non-R-PTP$\mu$ expressing carcinoma cells did, suggesting that expression of R-PTP$\mu$ arrested cell growth. To better understand the growth arrest induced by R-PTP$\mu$, we transfected the H-ras transformed lung epithelial cell line (MvLu-1-ras) with R-PTP$\mu$ (MvLu-1-ras/R-PTP$\mu$). Examination of growth factor receptor phosphorylation revealed significant inhibition of c-met and EGF-R. Furthermore, these cells underwent apoptosis in the absence of serum. Taken together the data demonstrate that the downregulation of R-PTP$\mu$ expression is an important step in neoplastic transformation of lung epithelial cells and that its presence can induce apoptosis and inhibit the signaling of c-met and EGF-R, two major growth factor receptors in lung carcinoma. In conclusion, the expression of R-PTP$\mu$ is inversely correlated with neoplastic transformation, growth and survival of tumor cells. ^

Activation of c -Met contributes to growth and metastasis of human colorectal carcinoma

c-Met is the protein tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF/SF) and mediates several normal cellular functions including proliferation, survival, and migration. Overexpression of c-Met correlates with progression and metastasis of human colorectal carcinoma (CRC). The goals of this study were to determine if overexpression of c-Met directly contributes to tumorigenicity and liver metastatic potential of colon cancer, and what are the critical pathways regulated by c-Met in this process. The studies used two colon tumor cell lines, KM12SM and KM20, which express high levels of constitutively active c-Met and are highly metastatic in nude mice. To examine the effects of c-Met overexpression, subclones of theses lines with reduced c-Met expression were obtained following transfection with a c-Met specific targeting ribozyme. Reduction of c-Met in KM12SM cells abolished liver metastases when cells were injected intrasplenically in an experimental metastasis assay. However, c-Met downregulation in theses clones was unstable. Three stable KM20 clones with a 25–35% reduction in c-Met protein levels but 60–90% reduction in basal c-Met autophosphorylation and kinase activity were obtained. While HGF increased c-Met kinase activity in the clones with reduced c-Met, the activity was less than that observed in parental or control transfected cells. Correlating with the reduction in c-Met kinase activity, subclones with reduced c-Met expression had significantly reduced in vitro growth rates, soft-agar colony forming abilities, and increased apoptosis. HGF/SF treatment did not affect anchorage-dependent growth or soft-agar colony forming abilities. Further, c-Met downregulation significantly impaired the ability of HGF/SF to induce migration. To examine the effects of reduced c-Met on tumor formation, parental and c-Met reduced KM20 cells were grown subcutaneously and intrahepatically in nude mice. c-Met downregulation delayed, but did not abolish growth at the subcutaneous site. When these cells were injected intrahepatically, both tumor incidences and size were significantly reduced. To further understand the molecular basis of c-Met in promoting tumor growth, the activation of several signaling intermediates that have been implicated in c-Met mediated growth, survival and migration were compared between KM20 parental cells and subclones with reduced c-Met expression levels. The expression and activity (as determined by phosphorylation) of AKT and Erk1/2 were unaltered. In contrast, Src kinase activity, as measured by immune complex kinase assay, was reduced 2–5 fold following c-Met downregulation. As Src has been implicated in growth, survival and migration, Src activation in c-Met overexpressing lines is likely contributing to the tumorigenic and metastatic capabilities of colon tumor cell lines that overexpress c-Met. Collectively, these results suggest that c-Met overexpression plays a causal role in the development of CRC liver metastases, and that c-Src and c-Met inhibitors may be of potential therapeutic benefit for late-stage colon cancer. ^