DL Cells Research Articles

Antiproliferative activity of Fe(II), Co(II), Ni(II), Cu(II), and Zn(II) complexes of dithiocarbamate: synthesis, structural characterization, and thermal studies.

Five new metal complexes of Fe(II) (1), Co(II) (2), Ni(II) (3), Cu(II) (4), and Zn(II) (5), derived from an N-cyclohexyl N-(3,4-dimethoxybenzyl) dithiocarbamate ligand, have been successfully synthesized and fully characterized by different analytical techniques i.e. elemental analyses, FT-IR, UV-Vis, 1H & 13C NMR, and HRMS. Furthermore, complexes 4 and 5 have been characterized by the SC-XRD technique. Complex 4 adopts a distorted square planar geometry around the Cu(II) center while complex 5 adopts a distorted tetrahedral geometry around the Zn(II) center. In addition, an eight-membered symmetric chair-like metallacycle ring containing two Zn(II) centers has also been found in complex 5. XRD data also show that complexes 4 and 5 are stabilized by various weak intermolecular hydrogen bonding interactions. The course of the thermal degradation of metal complexes 1-5 has been examined by TG-DTA data which revealed that metal sulphide formed as the final residue. Complexes 1-5 demonstrated concentration-dependent cytotoxicity and growth inhibition of DL tumor cells. Among the compounds, complexes 1, 4, and 5 showed significant cytotoxicity and induced a loss in the viability of DL cells. Therapy with complexes 1, 4, and 5 protects DL tumor-bearing animals from exacerbation of the disease, increases lifespan, and significantly improves the histopathological parameters of the vascularized organ, including preventing metastasis. Overall cytotoxicity assay results indicate that all complexes have remarkable cytotoxic potential in comparison with the free ligand.

Solvent-dependent crystallization and anti-cancer activities based on Ni(II) and Co(II) complexes of 1-picolinoyl-4-phenyl-3-thiosemicarbazide: Synthesis, crystal structure, and photoluminescence study

In search of alternative of platinum-based drugs for the treatment of cancer, lead to the development of other potential metallodrug of transition metal complexes. The efficacious and novel experimental content of this paper reports the synthesis of [Ni(Hppts)2].CHCl3 (1a), [Ni(Hppts)2].(CH3)2SO (1b) and [Co(Hppts)2] (2) complexes of 1-picolinoyl-4-phenyl-3-thiosemicarbazide (H2ppts). The synthesized complexes have been characterized by UV-vis., Infrared, and NMR spectrometry. Furthermore, complexes 1a and 1b were characterized by single-crystal X-ray diffraction data. Emission spectra show that, complex 1a exhibits higher fluorescence intensity as compared to that of ligand H2ppts and complex 2. The order of fluorescence intensity was found as complex 1a > complex 2 > H2ppts. Moreover, Complexes 1a and 1b are stabilized via various types of intermolecular interactions. The cytotoxicity of complexes 1a, 2, and ligand was evaluated against Dalton's Lymphoma cells using standard MTT Assay. The anticancer activity results showed that complex 1a significantly reduced cell viability in a dose-dependent manner, whereas H2ppts and complex 2 did not show significant reduction in cell viability of DL cells when compared with the control. The complex 1a IC50 value was determined to be around 40 µg/mL. The anti-tumor activity concludes that the complex 1a has high anti-neoplastic activity on DL cells at low doses.

Moringa oleifera L. leaf extract induces cell cycle arrest and mitochondrial apoptosis in Dalton's Lymphoma: An in vitro and in vivo study

Ethnopharmacological relevanceThe present work is based on a wide spectrum of evidences available from scientific literature which reflects nutritional and medicinal values of natural products such as plants and their extracts. Moringa oleifera is one such popular plant species amidst indigenous tribal communities which is frequently used to treat ailments such as piles, sore throat, eye and ear infections and even poisonous bites of tropical fauna such as insects or snakes. Furthermore decoction of leaf and bark was used to cure fever and cough. Evidences further reveal that Moringa oleifera L. (Family Moringaceae), is widely distributed not only over the Indian sub-continent, but also over Philippines, Central America, Saudi Arabia and the Caribbean Islands and have been traditionally used to treat cancers since ancient times. However, therapeutic effects of Moringa oleifera on Non-Hodgkin Lymphoma (NHL) are yet to be established. Aim of the studyThe study aims to investigate the anti-cancer effects of Moringa oleifera leaf extract against murine NHL Non-Hodgkin cells in vitro and in vivo. Material and methodsThe pharmacologically active compounds of Moringa oleifera leaf extract were identified by GC-HRMS analysis. Tests of Moringa oleifera leaf extract's cytotoxicity against DL cells were carried out using the MTT assay. Chromatin condensation along with other morphological alterations were visualized through Fluorescence microscopy. Changes in the mitochondrial membrane potential (ΔΨm), the cell cycle, and apoptosis were analysed through flow cytometer. We tried to identify proteins involved in apoptosis and cell cycle through Western blotting using BALB/c mice as a model organism. ResultsGC-HRMS study revealed that a methanol based leaf extract of Moringa oleifera (MOML) comprises of a variety of bioactive chemicals. Our results indicate that MOML successfully reduced the proliferation of DL cells by lowering ΔΨm, changing overall cell morphology. DL cells treated with MOML showed arrested cell cycle at the G2/M phase and substantially up-regulated the expression of p53 and p21. Elevated levels of Bax, Cyt-c, and Caspase-3 and lowered expression levels of Bcl-2 protein suggested induction of apoptosis. Mechanistically, the anticancer efficacy of MOML is attributed to MEK/ERK-mediated pathway inactivation in DL cells. It is also interesting to note that MOML-mediated inhibition of DL growth was accompanied by apoptosis induction and improvement in hematological parameters in DL-bearing mice. ConclusionOur finding suggested that MOML induces apoptosis and abrogates the growth of Dalton's lymphoma both in vitro and in vivo.

Evidence that PKCα inhibition in Dalton’s Lymphoma cells augments cell cycle arrest and mitochondrial-dependent apoptosis

Protein kinase Cα (PKCα), belonging to ser/thr protein kinase, perform various biological functions. Overexpression of PKCα has been observed in multiple human malignancies including lymphoma. However, the molecular pathogenesis and involvement of PKCα in Non-Hodgkin lymphoma (NHL) are not clearly understood. Hence, deciphering the role of PKCα in NHL management may provide a better therapeutic option. In the present study, we used selective pharmacological inhibitors Gö6976 and Ro320432 that potentially inhibit PKCα-mediated signaling in DL cells, resulting in the inhibition of cell growth and mitochondrial-dependent apoptosis. PKCα inhibition by these inhibitors also displays cell cycle arrest at the G1 phase and causes growth retardation of DL cells. Our results extended the mechanism of PKCα in NHL, and provided potential implications for its therapy.

Biochemical and mitochondrial membrane potential changes relating to betulinic acid-mediated anticancer activity in murine ascites Dalton's lymphoma

BackgroundBetulinic acid, a naturally occurring pentacyclic triterpenoid is found in a variety of plants. A range of biological properties including anticancer activity has been reported for betulinic acid. Changes in glutathione and some glutathione-related enzymes and mitochondrial membrane potential have not been explored relating to the anticancer activity of betulinic acid against murine ascites Dalton's lymphoma.The present study was undertaken to examine betulinic acid-mediated changes in glutathione and mitochondrial membrane potential in murine malignant ascites Dalton's lymphoma cells to identify their involvement as the possible mechanism in its anticancer activity. Method: The anticancer efficacy of betulinic acid was explored as a percentage increase in the life span (ILS) of the tumor-bearing mice. Betulinic acid-mediated changes in glutathione level, glutathione-related enzymes, succinate dehydrogenase activity, and mitochondrial membrane potential were determined in Dalton's lymphoma cells using well-established methods. In silico molecular docking study was done to know the binding affinity of betulinic acid with selected glutathione related enzymes and succinate dehydrogenase. Results: Betulinic acid significantly increased the life span of ascites Dalton's lymphoma-bearing hosts, thus exhibiting anticancer potential. The treatment caused a decrease in glutathione and glutathione-related enzymatic activities in DL cells. In silico molecular docking analysis revealed that betulinic acid illustrates good binding affinity with glutathione-related enzymes indicating its inhibitory potentials. Further, a decrease in mitochondrial membrane potential and succinate dehydrogenase activity was also noted after treatment. Conclusions: Betulinic acid-mediated decrease in glutathione in DL cells may hamper cellular antioxidant defense potential, which could play an important causative role in exerting its anticancer activity. Further, a concurrent decrease in mitochondrial membrane potential and succinate dehydrogenase activity is also important in its anticancer activity.

Unconventional formation of a 1D-chain of H-bonded water molecules in bipyridine-based supramolecular hexameric hosts of isostructural coordination compounds of Co(II) and Zn(II): Antiproliferative evaluation and theoretical studies

Two new isostructural coordination compounds of cobalt(II) and zinc(II) involving 2,2′-bipyridine and 2,5-pyridine dicarboxylate, viz. [Co(2,5-PDC)(bipy)2]·7H2O (1) and [Zn(2,5-PDC)(bipy)2]·7H2O (2) (bipy = 2,2′-bipyridine and 2,5-PDC = 2,5-pyridine dicarboxylate), have been synthesized in purely aqueous medium at room temperature. The compounds have been further characterized by single crystal X-ray diffraction, FT-IR, electronic spectroscopy, thermal and elemental analysis. Several non-covalent interactions, such as CH⋯O and anti-parallel π-stacking contacts, build up the supramolecular layered architectures of the crystal structures. Further analysis of the crystal structures reveals the self-assembled enclathration of a 1D H-bonded (H2O)8 chain into the supramolecular hexameric host cavity which brings rigidity to the crystal structures. Such unconventional enclathration of H-bonded water molecules in a metal–organic supramolecular host involving bipy is scarcely described in the literature. We have theoretically evaluated the strength of the anti-parallel π-stacking interactions that are crucial for the stabilization of the supramolecular dimers of the compounds. The influences of the metal coordination on the strength of the π-stacking interactions have been confirmed using DFT calculations and NCI plot computational tools. The antiproliferative activities of the compounds have been investigated in the DL cell line using MTT cell viability and apoptosis assays. The compounds significantly induce concentration dependent cell cytotoxicity and apoptosis in DL cells with negligible cytotoxicity in normal (PBMC) cells. A molecular docking study also reveals a strong interaction of the compounds with the active site of anti-apoptotic BCL proteins. Pharmacophore features of the structures of the compounds responsible for the biological activities were also generated to establish a structure activity relationship (SAR).

Antioxidant and Antiproliferative Activity of Selected Medicinal Plants of Lower Assam, India: An In Vitro and In Silico Study.

The use of medicinal plants for general wellbeing and disease treatment is a common practice among tribal communities of Kokrajhar districts of Assam. However, little works have been done to study the pharmacological aspect of the plants. The present study intends to study the antioxidant and antiproliferative properties of selected medicinal plants used by the tribal communities of the Kokrajhar district of Assam since ancient times. Five traditionally important medicinal plants, namely, Cassia fistula, Citrus grandis, Lindernia crustacea, Sacciolepis myosuroides, and Zingiber zerumbet were investigated for antioxidant, antiproliferative (cytotoxic) and apoptosis-inducing potential in the malignant cancer cell line. Phytochemical content, such as phenolic and flavonoid content, were estimated following standard protocol. The methanolic extract of plants was investigated following the phosphomolybdate method (TAC), FRAP, DPPH, ABTS, and TBARS assays. Antiproliferative activities of the plants were carried out by MTT assay in DL and PBMC cells. The apoptotic study was carried out following the acridine orange and ethidium bromide staining method and fluorescent microscopic imaging. Based on the significant (P≤0.05) high apoptotic inducing potential of the plant and to further dissect the molecular mode of action, including downstream biological action, major phytochemicals derived from L. crustacea were investigated for its prospective binding affinity with anti-apoptotic cancer target proteins. Antioxidant studies by FRAP, DPPH, ABTS, and TBARS assay revealed that all five plants contain considerable free radical scavenging activity. C. fistula showed the strongest free radical scavenging activity while the fruit peel extract of C. grandis showed poor activity. The overall antioxidant activities of plants such as TAC, FRAP, DPPH, ABTS, and TBARS may be arranged in decreasing activity as C. fistula > Z. zerumbet > L. crustacea > S. myosuroides > C. grandis. MTT based cell proliferation study showed that all the plants extract significantly (P≤0.05) inhibited cell viability with negligible cytotoxicity (~5-12%) in normal cells. Moreover, L. crustacea showed promising antiproliferative and apoptosis-inducing ability against Dalton's lymphoma. It is worth mentioning that the major bioactive compounds of the most potent plant extract, L. crustacea interacted with anti-apoptotic proteins (cancer target) with higher affinity and the results are compared with reference inhibitors. It is worth noting that these plants have the potential to consider for further scientific studies in different cell lines and animal models. Furthermore, isolation and characterization of bioactive compound(s) may promise the discovery of new and valuable drugs candidate to tackle various human diseases.

Energetically favorable anti-electrostatic hydrogen bonded cationic clusters in Ni(II) 3,5-dimethylpyrazole complexes: Anticancer evaluation and theoretical studies

Two new Ni(II) coordination complexes of pyrazole and acetato ligands, involving nitrate and chloride counter anions, viz. [Ni(Hdmpz)2(CH3COO)(H2O)3]NO3·2H2O (1) and [Ni(Hdmpz)2(CH3COO)(H2O)3]Cl (2) (where Hdmpz = 3,5-dimethylpyrazole), have been synthesized in purely aqueous medium at room temperature. The complexes have been characterized by elemental analysis, FT-IR, electronic spectroscopy, PXRD, TGA and single crystal X-ray diffraction. The H-bonds established between the isostructural cationic [Ni(Hdmpz)2(CH3COO)(H2O)3]+ moieties of both complexes can be classified as anti-electrostatic H-bonds (AEHBs) and these have been studied using DFT calculations. Remarkably, in one case these AEHBs exhibit favorable interaction energies, thus indicating that the attractive nature of the H-bonds is able to compensate the pure electrostatic repulsion. The analyses of the cell viability results for the DL cell line show a significant concentration dependent decrease by compounds 1 and 2 with a negligible cytotoxic effect observed against normal cells (PBMC) in comparison to the cancer cells (DL). The cytotoxic activity of these compounds in DL cells occurs via apoptosis. Molecular docking and SAR analysis reveal that the compounds possess high binding affinities against most common cancer target proteins.

Selenium nanoparticles induce suppressed function of tumor associated macrophages and inhibit Dalton's lymphoma proliferation

Selenium Nanoparticle (SeNPs) is reported that it enhances and maintains optimal immune during infection and malignancies. To this end, we examined the role of selenium on TAMS whose anti-tumor function suppressed which favor tumor progression. BALB/c (H2d) strain of mice non-Hodgkin type of Dalton's cell line was used to check the role of carboxlic group induced, synthesized SeNPs on TAMs. Screening of IC50 value was done primarily trypen blue exclusion assay and 50% proliferation of DL cells inhibited 40ng/ml to 50ng/. Treatment also decreases ΔΨm, fragmentation of DNA of DL cells and arrest cells cycle in G1/G0 phage. Untreated TAMs cells showing suppressed expression of ROS, adhesion, phagocytosis, fusion and receptor profiling such as ICAM-1, CD47, CD172α. Which was induced more as compare to untreated group. SeNPs have potential to induce the anti-tumor function of TAMs whose anti-tumor function down-regulated pliable shifted towards tumor progression. It decreased the proliferation of DL cell by inducing apoptosis. Therefore, the synthesized SeNPs could be used for imaging diagnosis and cancer therapy which must be cost effective with negligible side effects shifted towards tumor progression. It decreased the proliferation of DL cell by inducing apoptosis.

Downregulation of STAT3 phosphorylation enhances tumoricidal effect of IL-15-activated dendritic cell against doxorubicin-resistant lymphoma and leukemia via TNF-α

Although disputed by some, increasing evidence suggests that TNF-α synergies with traditional chemotherapeutic drugs to exert a heightened antitumor effect. The present study investigated the antitumor efficacy of recombinant IL-15 in combination with the STAT3 inhibitor cucurbitacin-I in a doxorubicin-resistant murine lymphoma model. The significance of the work is to understand and design effective strategies in doxorubicin resistant lymphomas. TNF-α is downregulated in dendritic cells from mice with Dalton's lymphoma and shows an inverse relationship with disease progression. Doxorubicin-resistant DL cells have elevated levels of Bcl-2 and Mcl-1 and increased phosphorylation of STAT3. These cells are refractory to dendritic cell derived TNF-α. Doxorubicin resistant Dalton's lymphoma is susceptible to dendritic cell derived TNF-α upon stimulation with the STAT3 inhibitor cucurbitacin-I, which downregulates STAT3 and other survival molecules. The combined treatment of low dose of cucurbitacin-I and rIL-15 is ineffective in mice with doxorubicin resistant Dalton's lymphoma, but a similar therapy prolongs the survival of mice transplanted with parental Dalton's lymphoma. Doxorubicin resistant Dalton's lymphoma responds to therapy with high doses of cucurbitacin-I and rIL-15. Dendritic cell derived from mice responded positively to the therapy and regained their tumoricidal properties with respect to growth inhibition and killing of DL tumor cells. Similar to DL, DC derived from CML patients are impaired in TNF-α expression and are unable to restrict the growth of drug-resistant lymphoma and leukemia cells. This combination approach could be used as a new therapeutic strategy for aggressive and highly metastatic doxorubicin resistant lymphoma.

Abstract 4966: Establishment and characterization of a paired human non-small cell lung cancer cell lines from primary cancer and metastasis lymph node

Abstract Objective: Lung cancer has become one of the leading causes of cancer-related mortality in the world. Metastasis remains the overwhelming causes of death for lung cancer patients. 80-90% of lung cancer deaths are caused by metastasis. However, an ideal in vitro model to explore the regulation and the molecules that control the lymph node metastasis of lung cancer is required. Methods: A gene expression profiling for both of the cell lines were generated by microarray. Further investigations were carried out in the lung cancer cell lines and paired tissues by migration and invasion assays and immunohistochemistry. Results: Firstly, we established a paired human lung adenocarcinoma cell lines from primary tumor site (named DC cells) and metastatic paratracheal lymph nodes (named DL cells). The two cell lines have different biological characteristics which DC cells grow clump easily and DL cells grow more scattered. The doubling time of DC and DL cell lines were 32.6h and 45.2h, respectively. The detection of the human D1S80 and D17S30 VNTR locus verified the same individual for DC and DL cell lines. Secondary, Microarray analysis revealed 232 upregulated probe sets and 739 downregulated probe sets in DL cell line compared to DC cell line. Compared to DC cells, the transcriptional expression of 27 genes were dramatically increased (p < 0.05), including AIM2, IGFBP4, IL6 and IL6R in DL cells, while expression of 55 genes was clearly decreased (p < 0.05), including FGF13, FGF19, IL1A, IL1B, MCAM, MMP15, MMP7, and TGFB2 by real-time PCR analysis. Furthermore, we selected 32 genes and screened the expression of those genes in 10-paired human NSCLC primary lung cancer tissues and matched metastasis lymph nodes tissues by real-time PCR. The results showed: 8 out of 10 metastasis lymph node tissues got up-regulated of RGS7, VNN2, and 7 out of 10 metastasis lymph node tissues got up-regulated of PECAM1, VNN1, CLIC2 expression. On the other hand, 8 of 10 metastasis lymph node tissues got down-regulated expression of CEACAM6, and 7 out of 10 got down-regulated expression of PIAS3. At mean time, none of metastasis lymph node tissues got decreased expression of TNFRSF9, which only was seen in DL cells. The further study in other lung cancer cell lines indicated that CLIC2, PECAM1, RAB38, BCL9 and RGS7 genes influenc lung cancer cell migration ability in vitro. And these genes were also analyzed in more lung cancer tissues by immunohistochemistry staining. Conclusion: We established a paired human lung adenocarcinoma cell lines from primary tumor site and metastatic paratracheal lymph nodes. The CLIC2, PECAM1, RAB38, BCL9 and RGS7 genes involve in the lung cancer metastasis. Note: This abstract was not presented at the meeting. Citation Format: Jun Chen, Min Wang, Lingling Zu, Yongwen Li, Weiqiang Wang,Ying Li, Hongyu Liu. Establishment and characterization of a paired human non-small cell lung cancer cell lines from primary cancer and metastasis lymph node. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4966. doi:10.1158/1538-7445.AM2014-4966

Structural and biochemical changes in mitochondria after cisplatin treatment of Dalton’s lymphoma-bearing mice

Cisplatin treatment of tumor-bearing mice and analysis of ultrastructural features of mitochondria in the kidney and Dalton’s lymphoma cells showed the appearance of more roundish mitochondria with thickened membranes. It also caused the reduction in the number and irregularity in the shape of cristae and formation of vacuoles in the mitochondria. After cisplatin treatment, decreased level of protein, succinate dehydrogenase activity, and increased level of lipid peroxidation were noted in Dalton’s lymphoma tumor cells and kidney. Cisplatin-mediated decrease in SDH activity, GSH level and an increase in LPO in the mitochondria of kidney could play an important role to produce nephrotoxicity. However, in DL cells, decrease in cellular GSH could be noteworthy than mt-GSH, along with decrease in SDH activity and increase in LPO in the cisplatin-mediated anticancer activity. These changes could play an important role to produce both the cisplatin-mediated effects i.e. anticancer activity and nephrotoxicity. Cisplatin-induced biochemical and ultrastructural changes in mitochondria after cisplatin treatment should be an important factor in the development of biochemical injury in mitochondria and affecting the overall metabolism in the cells. The findings from the present studies indicate multilevel effect of cisplatin in the cells and do support the earlier view that mitochondria could be a critical target in cisplatin-mediated anticancer activity and toxicity in the hosts.

Overexpression of Interleukin-13 in a Murine T-Cell Lymphoma: A Possible Factor of DL-Induced Immunosuppression and Tumor Progression

Interleukin-13 (IL-13) is a TH2 cytokine that plays a crucial role in the pathophysiology of tumors and favor tumor growth and recurrence by negative regulation of tumor immunosurveillance. In the present investigation, we have determined the IL-13 level in the serum and ascitic fluid of the DL-bearing host and the possible source of IL-13 in the ascitic fluid. IL-13 level was elevated in serum and ascitic fluid of host bearing a transplantable T-cell lymphoma. DL cells as well as tumor-associated macrophages express and secrete IL-13 in the milieu, which provide further insight for DL-induced immunosuppression in a tumor-bearing host.

Regression of Dalton’s lymphoma in vivo via decline in lactate dehydrogenase and induction of apoptosis by a ruthenium(II)-complex containing 4-carboxy N-ethylbenzamide as ligand

A novel ruthenium(II)-complex containing 4-carboxy N-ethylbenzamide (Ru(II)-CNEB) was found to interact with and inhibit M4-lactate dehydrogenase (M4-LDH), a tumor growth supportive enzyme, at the tissue level. The present article describes modulation of M4-LDH by this compound in a T-cell lymphoma (Dalton's Lymphoma: DL) vis a vis regression of the tumor in vivo. The compound showed a dose dependent cytotoxicity to DL cells in vitro. When a non toxic dose (10 mg/kg bw i.p.) of Ru(II)-CNEB was administered to DL bearing mice, it also produced a significant decline in DL cell viability in vivo. The DL cells from Ru(II)-CNEB treated DL mice showed a significant decline in the level of M4-LDH with a concomitant release of this protein in the cell free ascitic fluid. A significant increase of nuclear DNA fragmentation in DL cells from Ru(II)-CNEB treated DL mice also coincided with the release of mitochondrial cytochrome c in those DL cells. Importantly, neither blood based biochemical markers of liver damage nor the normal patterns of LDH isozymes in other tissues were affected due to the treatment of DL mice with the compound. These results were also comparable with the effects of cisplatin (an anticancer drug) observed simultaneously on DL mice. The findings suggest that Ru(II)-CNEB is able to regress Dalton's lymphoma in vivo via declining M4-LDH and inducing mitochondrial dysfunction-apoptosis pathway without producing any toxicity to the normal tissues.

Real-Time In Vivo Biodistribution of Multipotent Adult Progenitor Cells (MAPC): Role of the Immune System in MAPC Resistance in Non-Transplanted and Bone Marrow Transplanted Mice.

MAPC are non-hematopoietic stem cells derived from adult BM with the potential for a wide differentiation pattern in vitro and in vivo. MAPCs are MHC class I and thus may be a target of natural killer (NK) cell mediated elimination in the syngeneic setting. To determine whether MAPC are susceptible targets for NK mediated killing, splenocytes from poly I:C (an inducer of NK activity) treated C57BL/6 mice were mixed with Yac-1 (H2a; a NK sensitive target) or MAPC (from C57BL/6J-rosa26) in a chromium release assay. Effector:target ratios indicated that MAPC were susceptible to NK lysis albeit less so than Yac-1 cells. To assess in vivo immune responses to MAPC, we infused MAPC into mice with various degrees of T-, B-, and NK- cell immune competence. To follow biodistribution of MAPC in live animals with whole body imaging (WBI), we labeled MAPC with red fluorescent protein DsRed2 and luciferase, using Sleeping Beauty transposons. MAPC (106) were co-nucleofected (Amaxa) with 5mcg of each pT/CAGGS-DsRed2 and pT/CAGGS-Luciferase and an SB transposase-encoding plasmid (p/CMV-HSB2) at a 1:50 ratio. Selected double transgenic MAPC (MAPC DL) clones were euploid, and maintained their characteristic trilineage differentiation. MAPC DL (106) were injected IV into cohorts (n=5–6) of adult C57BL/6 (B6), Rag2−/− (T- and B-cell deficient) and B6 Rag2/IL-2Rgc (T-, B- and NK deficient mice). Additional cohorts of B6 and Rag2−/− were given anti-NK1.1 mAb 2x/wk to deplete NK cells. In B6 mice, MAPC DL were detected on d4 but not d14 or d30. In Rag2−/− mice, MAPC DL were detected throughout the 30d period. NK depletion did not substantially increase MAPC DL number in B6 mice. However, in Rag2/IL-2Rgc mice MAPC DL were persistent and in 50% of mice they increased in number from d4‡d30. Post-mortem analysis revealed MAPC DL cells in all but B6 wild type mice: Rag2/IL-2Rgc ≥ Rag2−/− with NK depletion>> Rag2−/−. These data suggest that endogenous NK cells and T cells resist MAPC DL. Interestingly, in vitro studies indicate that MAPCs suppress an allogeneic mixed lymphocyte reaction (MLR) culture. Therefore, the T cell resistance to MAPC may be due to an immune response generated to the multiple foreign reporter proteins expressed by these cells. Since MAPCs may be useful as cellular therapies for the treatment of regimen-related toxicity, studies were performed in which B10.BR mice were lethally irradiated (TBI) and given B6 BM ± MAPC DL (106). MAPC DL were seen in the chest, abdomen, face, and paws on d4, d7, d10 and d28 at high numbers suggesting that TBI conditioning overcomes both NK and T cell mediated resistance resuting in a widespread homing/migration of MAPC. These data are the first to illustrate the immune responses to MAPCs and indicate that TBI conditioning may be advantageous in the long-term survival and widespread homing of MAPCs.

Differentiation and Maturation of Equine Erythroid Cells from Peripheral Blood in a Two Phase Liquid Culture System

The differentiation and maturation of erythroid cells from equine peripheral blood in a two-phase liquid culture system were researched based on morphological, hemoglobin and antigenic aspects. 1) By this culture system, the blastic cells arose sequentially basophilic erythroblasts, polychromatic erythroblasts, orthochromatic erythroblasts and denucleated erythroid cells. Orthochromatic erythroblasts were observed on day 3 of the 1st phase, and reached 33% of the total cells on day 5 of the 1st phase, followed by the appearance of denucleated erythroid cells. On day 7 of the 2nd phase, approximately 70% of cells were denucleated erythroid cells. 2) Hemoglobin containing cells were observed on day 1 of the 1st phase and exceeded 92% of the total cells on day 3 of the 2nd phase. 3) Blood group antigens were firstly observed on day 3 of the 1st phase. On day 7 of the 2nd phase, the rates of Aa, Ca, Dk, Dl, Qa, Qb and Qc positive cells were 92, 78, 52, 51, 77, 77 and 75%, respectively. These results suggest that Aa Ca, Qa, Qb and Qc antigens are expressed on orthochromatic erythroblasts, but that Dk and Dl antigens are expressed on denucleated erythroid cells.

Endotoxin-induced protein phosphorylation in macrophages is modulated by tumor cells

Tumor cells are known to modulate the antitumor functions of endotoxin or cytokine-stimulated macrophages, however, their mechanism of action is not known. We have recently shown that Daltons fn2 fn2 DL, Daltons lymphoma; DLL, DL cell lysate; LPS, lipopolysaccharide; PKC, protein kinase C; PTK, protein tyrosine kinase; PEC, peritoneal exudate cells; PBS, phosphate buffer saline; NO, nitric oxide; IL, interleukin; TNF, tumor necrosis factor. lymphoma (DL, a murine spontaneous T cell lymphoma) alter the activation of macrophages by lipopolysaccharide (LPS). In this investigation, the effects of DL cells on protein kinase C (PKC) activity, calcium uptake and protein tyrosine phosphorylation in murine peritoneal macrophages was studied. Treatment of macrophages with LPS resulted in the translocation of PKC from cytosol to the membrane fraction. Incubation of macrophages with DL cells or DL cell lysate (DLL) resulted in a significant decrease in the PKC activity in membrane fraction compared to the LPS-treated macrophages incubated without DL cells or DLL. DL cells were also found to inhibit the accumulation and influx of calcium in the macrophages in response to LPS. On the other hand, DL cells augmented the protein tyrosine phosphorylation in murine macrophages. Only viable DL cells were found to increase the protein tyrosine phosphorylation whereas DLL did not have any effect on protein tyrosine phosphorylation in macrophages. These results thus suggest that tumor cells and\\or their products can differentially effect the phosphorylation of different proteins involved in cell signalling.

Cloning of the human orphan receptor germ cell nuclear factor/retinoid receptor-related testis-associated receptor and its differential regulation during embryonal carcinoma cell differentiation.

We have cloned a cDNA encoding the full-length coding region of the human homologue of the germ cell nuclear factor (GCNF)/retinoid receptor-related testis-associated receptor (RTR), from a human testis cDNA library. The amino acid sequence of human GCNF/RTR is highly homologous to that of the mouse GCNF/RTR. The largest difference between the two homologues is a 15 amino acid deletion in the human GCNF/RTR at amino acid 47. The GCNF/RTR gene was localized on human chromosome 9. Northern blot analysis using poly(A)+ RNA from different human tissues showed that GCNF/RTR mRNA is most abundantly expressed in the testis. GCNF/RTR was also highly expressed in embryonic stem cells and embryonal carcinoma cells but repressed in its differentiated derivatives. Induction of differentiation of mouse embryonal carcinoma F9 cells and human embryonal carcinoma NTERA-2 clone D1 (NT2/D1) cells by all-trans retinoic acid was accompanied by a down-regulation of GCNF/RTR. Our observations suggest that GCNF/RTR plays a role in the control of gene expression in early embryogenesis and during spermatogenesis.

Alteration in IL-1 and arginase activity of tumor-associated macrophages: a role in the promotion of tumor growth

The present investigations were undertaken to study whether the macrophages associated with Dalton's lymphoma (DLAM), a spontaneous T-cell lymphoma, are modulated for the secretion of tumor growth-promoting factors in ascites. The DL ascitic fluid (DLAF) and the culture supernatants of DLAM harvested from mid and late tumor-bearing stages, but not early tumor-bearing stage, enhanced the proliferation of DL cells in vitro. These observations indicate that DLAF contains certain DL growth-promoting substances and at least some of them are secreted by DLAM. The DLAM obtained from mid and late tumor-bearing stages showed enhanced IL-1 production and arginase activity, with a concomitant decline in the RNI production, which could be implicated in the DLAM-mediated promotion of tumor growth.

Learning algorithms for oscillatory networks with gap junctions and membrane currents

One of the most important problems for studying neural network models is the adjustment of parametas. Here we show how to formulate the problem as the minimization of the dilferslee betwen two limit cycles. The backprop%ation method for leaming algorithms is described .ss the application of grsdient descent to an eror fundion that computes this difference. A mathematical formulation is given that is applicable to any type of network model, and is applied to several models. For example, when learning in a network in which dl cells have a common, adjustable, bias current, the value of the bias ie adjustcd at a rate proportional to the difference between the sum of the target outputs and the sum of the actual outputs. When learning in a network of n cells where a target output is given for every cell. the learning algorithm splits into R indepndent leaming algorithms, one per cell. For networks containing gap junctions, a gap junction is modelled D conductance times the potential difference between the two adjacent cells. The requirement that a conductme g munt be positive is enionzed by replacing g by a functbn pos(g*) whose value is always positive, for example cxp(O.lg*), and deriving an algorithm that adjusts the parameta g* in place of g. When target output is specified for every cell in a network with gap junctions. the learning algorithm splits into fewer independent componeds, one for each gap-mnneaed subset of the network. The lemming algorithm for II gspmnneeted set of cells cannot be paralklized further. As a find example, a leaming algwithm is derived for a mutually inhibitory twc- cell network in which each cell has a membrane current. This generalized approach to backpropagation allows one to derive a learning algorithm for alntoat any model neural network given in tem of differmtid equations. It will be an essential tool for adjusting parmetem in small but complex network models.