Reduced Thymidine Uptake Research Articles

Abstract 1796: Significant in-vitro anti-myeloma activity of a novel Akt inhibitor MK2206

Abstract Background: Cytokine stimulated signaling pathways contribute to multiple myeloma (MM) disease progression and in acquired resistance to current treatment options making MM an incurable malignancy. The PI3K/Akt/mTOR pathway is up-regulated in MM and appears to be critical for cell proliferation and blocking this pathway might have profound impact on disease progression. In addition, this pathway also has been found to cross talk with other signal transduction pathways preventing drug-induced apoptosis. Methods: MK2206 was obtained from Merck Inc. MTT assay was performed to study drug induced cytotoxicity and thymidine uptake was used as a measure to study differences in proliferation. Flow cytometry using Annexin V-FITC and propidium iodide (PI) was used to measure drug induced apoptosis in cell lines and patient cells. In addition, apo-2.7 was also used to measure apoptosis in patient cells. Results: MK2206 treatment led to dose and time dependent decrease in cell viability as observed by MTT assay with an IC50 range of 1-5µM on most MM cell lines tested. Most of the cytotoxicity was observed by 48hours with minimal increase in cytotoxicity beyond that point. The drug was able to induce cytotoxicity in myeloma cell lines when cells were cultured with IGF, IL6, or VEGF with minimal increase in IC50 values. MK2206 also inhibited MM cell proliferation as observed by reduced thymidine uptake. This reduction in proliferation was also observed when MM cells were co-cultured with bone marrow stromal cells. When myeloma cell lines were treated with the drug for various time points, percentage of apoptotic cells increased with time of incubation with the drug as observed by flow cytometry and annexin V-FITC/PI staining. There was also a time dependent increase in caspase cleavage observed indicating that MK2206 might elicit its functions by inducing caspase dependent pathways. Further studies are being carried out to understand the importance of caspase dependent pathways for the success of anti-MM effects of MK2206. Also, MK2206 was able to induce apoptosis in patient derived primary cells, though with varying degrees of response observed between patients. Examining signaling pathways by immunoblotting demonstrated clear down regulation of pAkt and Bcl-xl levels post treatment with the drug with minimal decrease in proteins associated with other signaling pathways, like pSTAT3 and pERK. Conclusions: Taken together, our studies demonstrate significant anti-MM activity of MK2206 in-vitro. We are currently testing MK2206 in combination with known inhibitors of the other important signaling pathways implicated in MM disease biology as well as in-vivo experiments in mouse models. Performing these experiments will further validate the efficacy of MK2206 as an anti-MM agent and form the basis for it to be taken up for clinical evaluation either as a single agent or in combination with other agent(s). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1796.

Vitamin C enters mouse T cells as dehydroascorbic acid in vitro and does not recapitulate in vivo vitamin C effects

Vitamin C is an essential micronutrient, which has been implicated in various biological processes, including immune response. In fact, in vivo administration of vitamin C modulates T cell proliferation and cytokine secretion. In this study, we analyzed the mechanism by which mouse T cells take up vitamin C, and whether this uptake directly affected T cell functions. T cells internalized more vitamin C when they were activated, due to enhanced glucose transporter (GLUT)-1 and GLUT-3 expression that persisted up to 48 h after activation. Blocking oxidation of ascorbic acid (the reduced form of vitamin C) in the culture medium with 1,4-dithio-threitol (DTT) almost completely inhibited the enhanced vitamin C uptake. The presence of vitamin C at low concentrations during in vitro T cell activation did not affect proliferation or cytokine secretion (IFN- γ, TNF- α, or IL-4) in response to PMA/ionomycin. In contrast, high concentrations (0.25–0.5 mM) of vitamin C lowered cell viability, reduced thymidine uptake, and decreased cytokine secretion. In conclusion, activated T cells upregulated GLUT-1 and -3 to increase vitamin C uptake. They took up vitamin C mostly in its oxidized form, rarely in its reduced form. Application of vitamin C to T cells in vitro did not recapitulate previously reported in vivo responses to vitamin C, suggesting that in vivo, vitamin C modulates T cells indirectly through other components of the microenvironment.

Differential control of mesangial cell proliferation by interferon-gamma

Rat mesangial cells were shown to be sensitive to recombinant interferon-gamma (IFN-gamma). IFN-gamma reduced thymidine uptake by these cells and inhibited cell proliferation. Incubation of the cells with 1000 U/ml IFN-gamma decreased thymidine uptake by up to 64% and cell numbers were decreased by 17%. The effects of IFN-gamma were dose and time dependent and were partially reversible by the anti-IFN-gamma monoclonal antibody DB-1. This lymphokine did not reduce incorporation of RNA and protein precursors however. Measurements of 3H-uridine and 3H-leucine incorporation indicated significant increases in RNA and protein synthesis (37% and 45%, respectively) on a per cell basis. The mitogenic effects of IL-1 and platelet-derived growth factor (PDGF) were also susceptible to IFN-gamma-mediated inhibition but the mitogenic response to epidermal growth factor (EGF) was much less sensitive. We conclude that while IFN-gamma may act to modulate the mitogenic signals provided by some factors such as IL-1 and PDGF, the response to EGF appears to be unaffected.

Doxycycline Alters Vascular Smooth Muscle Cell Adhesion, Migration, and Reorganization of Fibrillar Collagen Matrices

Remodeling of injured blood vessels is dependent on smooth muscle cells and matrix metalloproteinase activity. Doxycycline is a broad spectrum matrix metalloproteinase inhibitor that is under investigation for the treatment of acute coronary syndromes and aneurysms. In the present study, we examine the mechanisms by which doxycycline inhibits smooth muscle cell responses using a series of in vitro assays that mimic critical steps in pathological vascular remodeling. Doxycycline treatment dramatically increased smooth muscle cell adhesion to the substrate, as evidenced by interference reflection microscopy and immunostaining for paxillin and phosphotyrosine. Cell aggregation was also potentiated after treatment with doxycycline. Treatment with 104 mumol/L doxycycline reduced thymidine uptake by 58% compared with untreated cells (P < 0.05) and inhibited closure of a scrape wound made in a smooth muscle cell monolayer by 20% (P < 0.05). Contraction of a three-dimensional collagen gel was used as an in vitro model for constrictive vessel remodeling, demonstrating that treatment with 416 mumol/L doxycycline for 12 hours inhibited collagen gel remodeling by 37% relative to control (P < 0.05). In conclusion, we have shown that doxycycline treatment leads to dramatically increased smooth muscle cell adhesion, which in turn might limit responses in pathological vascular remodeling.

Effect of PGE1 on hypoxia or hypoxia/reoxygenation-treated human vascular endothelial cells

The aim of this study was to investigate about endothelial cell injury caused by hypoxia or hypoxia/reoxygenation and the effect of PGE1 on the injury using human umbilical vein endothelial cells (HUVEC) .Under hypoxia, HUVEC shrank and peeled from dish, accompanied with reduced thymidine uptake, induced DNA fragmentation, and increased caspase-3 activity. On the other hand, under reoxygenation after hypoxia, HUVEC restarted proliferation as a result of decreasing caspase-3 activity in a time dependent manner.When PGE1 was added prior to hypoxia, the number of living cells got fewer and caspase-3 activity was potentiated without changing of cAMP levels. In contrast, the addition of PGE1 just before reoxygenation, increased the number of living cells and decreased caspase-3 activity with increasing cAMP levels. Under neither hypoxia nor reoxygenation, the gene expression of any prostaglandin EP receptors was not observed. In conclusion, PGE1 showed opposite effects to be pro-apoptotic under hypoxia, and to be anti-apoptotic under reoxygenation, not through EP receptors.

Desferioxamine increases iron depletion and apoptosis induced by ara-C of human myeloid leukaemic cells.

We investigated whether changes in iron metabolism and the transferrin receptor (TRF-R) expression were involved in the antileukaemic effects of arabinoside cytosine (ara-C). Treatment with 100 nM ara-C for 48h reduced thymidine uptake and increased the surface expression of the TRF-R on leukaemic blasts derived from 13/16 (81%) patients and on the HL-60 and U-937 cell lines. Whereas intracellular non-haem iron was strongly depleted 24 h after ara-C addition, TRF-R up-regulation and recovery of intracellular non-haem iron concentration occurred together after a longer exposure of the cultured cells to the drug. Since iron is an essential regulator of cell proliferation we have evaluated the effects of the combination between ara-C and the iron chelator desferioxamine (DSF) on the growth of HL-60 and U-937 cells. We found that desferioxamine strongly potentiated the effects of ara-C on leukaemic cell growth inhibition and apoptosis. This is the first report of a positive interaction between ara-C and an iron chelator in terms of antileukaemic effects.

Effects of integrins on proliferation and apoptosis of renal epithelial cells after acute injury

We sought to determine the importance of integrins for recovery after acute tubular injury and to investigate the signal transduction pathways for integrin effects on cell cycle regulation involving proliferation and apoptosis. Primary cultures of rat renal proximal tubule epithelial cells were exposed to a superoxide-generating system to induce injury in the absence of overt necrosis. Integrin function was antagonized by the integrin recognition sequence tetrapeptide Gly-Arg-Gly-Asp (GRGD) or monoclonal antibody to beta 1-integrin. Injured cells had reduced thymidine uptake compared with normal cells. The presence of GRGD during recovery from injury caused a further 44% reduction in DNA synthesis but did not affect DNA synthesis in normal cells. Injured cells had an increased proportion of apoptosis that was further accentuated by exposed to GRGD during recovery. Integrin antagonism also stimulated apoptosis in uninjured cells. To investigate signal transduction mechanisms for this effect of integrins, inhibitors and activators of protein tyrosine kinase (PTK) and protein kinase C (PKC) were evaluated. Activation of PKC stimulated cellular proliferation, whereas inhibitors of PKC and PTK had no significant effect. Genistein, a PTK inhibitor, induced apoptosis in normal cells, mimicking the effect of integrin inhibition. On the other hand, PMA, an activator of PKC, prevented cells from becoming apoptotic when exposed to injury plus GRGD. The phosphorylation status of intracellular proteins was evaluated by immunoblotting with antiphosphotyrosine antibody. A similar pattern of decreased phosphorylation was observed after either integrin inhibition, injury, both, or PTK inhibition. These findings suggest that kinase cascades are involved in the effects of integrins on renal epithelial cell proliferation and apoptosis. After injury, an interaction between cells and the extracellular matrix is required for cells to proliferate and contribute to repair rather than to enter an apoptotic pathway.

Effects of natriuretic peptides on vascular smooth-muscle cells derived from different vascular beds

1. 1. This study explored the hypothesis that atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-natriuretic peptide (CNP) have differing antiproliferative and antihypertrophic effects on pulmonary artery (PA) and thoracic aorta (TA) smooth-muscle cells (SMCs). 2. 2. Cultured cells were exposed to 5% fetal calf serum (FCS) and angiotensin II (A-II) to induce DNA and protein synthesis, respectively. 3. 3. ANP (10 −7 M) significantly reduced thymidine uptake in TA by 31%±2% ( P⩽0.01) but not in PA ( P⩾0.05). 4. 4. In parallel experiments, BNP (10 −7 M) significantly reduced thymidine uptake in TA (−22%±5%, P⩽0.01), but not in PA cells ( P⩾0.05). 5. 5. CNP (10 −7 M) did not significantly alter thymidine uptake in TA cells exposed to FCS, but it did significantly reduce uptake in PA (−28.5%±4%) 2( P⩽0.05). 6. 6. Blunting by ANP (10 −7 M) of the A-II (10 −8 M)-induced increase in protein synthesis was significantly greater in PA than in TA cells. 7. 7. However, BNP and CNP (10 −7 M) exerted similar antihypertrophic effects on TA and PA cells exposed to A-II. 8. 8. The antiproliferative effects of BNP and ANP exceed those of CNP in TA SMCs, but CNP appears to be the most effective antiproliferative agent in PA SMCs. In addition, PA-derived SMCs are more sensitive to the antihypertrophic effects of ANP than TA-derived cells, suggesting phenotypic differences. The findings indicate that the natriuretic peptides may play complementary roles in modulating SMC proliferation and protein synthesis.

The effect of folate analogues on the thymidine utilization by human and rat marrow cells and the effect on the deoxyuridine suppression test.

Summary The deoxyuridine (dU) suppression test assesses the capacity of marrow cells or activated lymphocytes to convert exogenous dU into thymidine. In addition, the effect of the added cobalamin or folate on the result is used to determine the nature of the deficiency in megaloblastic marrows. This study shows that folates have a considerable effect on the uptake of labelled thymidine by marrows from patients with megaloblastic anaemia in the absence of added dU. Less thymidine was taken up by megaloblastic marrow cells if they were first incubated with 5-formyltetrahydrofolate, 10-formyltetrahydrofolate or tetrahydrofolate. 5-Methyltetrahydrofolate increased thymidine uptake in cobalamin-deficient marrows and reduced thymidine uptake in folate-deficient marrows. These results could be explained if added folates facilitated utilization of endogenous dU. However, the addition of folates or cobalamin did not affect the validity of a dU suppression test with human marrow. More marked changes were present on the addition of folates to rat marrow cells both from control and nitrous oxidetreated animals.

Mitogenic responsiveness of mouse, rat and human lymphocytes to Staphylococcus aureus cell wall, teichoic acid, and peptidoglycan.

The mitogenic activity of Staphylococcus aureus peptidoglycan (PG), teichoic acid (TA) and cell wall (CW, a PG-TA complex) for mouse, rat and human lymphocytes was determined by measuring DNA synthesis. At optimal concentrations PG was the most potent mitogen, inducing maximum stimulation of mouse spleen lymphocytes on day 2 of culture, rat spleen lymphocytes on day 3, rat lymph node cells on day 2, human peripheral blood lymphocytes on day 5, and human cord blood lymphocytes on day 4. CW was less mitogenic than PG for mouse splenocytes and human peripheral and cord blood lymphocytes, with maximum stimulation occurring on days 2, 6 and 5, respectively. CW was not mitogenic for rat lymphocytes, and TA was not mitogenic for mouse, rat and human lymphocytes. At high concentrations CW and TA were cytotoxic, as indicated by markedly reduced thymidine uptake and low viability. PG was not toxic to lymphocytes. Our results suggest that TA when bound to PG can at high concentrations mask the mitogenic activity of PG and render CW cytotoxic.

Humoral and cellular immunity in asthma

Parameters of humoral and cellular immunity have been measured in 91 asthmatic patients. Mean serum levels of IgG and IgE were raised. IgG levels were higher in those with a family history of asthma. IgE levels were higher in those with a past history of atopic eczema, but intrinsic and extrinsic asthma could not be differentiated on the basis of IgE levels. Thirteen of 74 patients failed to respond to tetanus immunization, while only 1 failed to respond to Salmonella typhi H antigen. Tetanus nonresponders had a raised mean serum IgA level, reduced spontaneous lymphocyte tritiated thymidine uptake, and reduced thymidine uptake in fetal calf serum. Eight of 87 patients failed to mount delayed hypersensitivity reactions to a battery of five intradermal antigens. The tritiated thymidine uptake of lymphocytes stimulated with phytohemagglutinin was normal in autologous serum, but reduced in fetal calf serum. The data support the hypothesis that asthma may be associated with immunodeficiency states.

Disturbances in developmental pathways leading to a neurological disorder of genetic origin, “leaner,” in mice

A linkage relationship between the leaner gene, la, and esterase gene, El, was described. The recombination frequency between these genes was found to be 3.84%. Developmental pathways leading to neurological disorders in the leaner mutant were investigated. It was found that the thymidine uptake by the brain, liver and spleen of the mutant mice was significantly lower than that of their normal sibs. There was a good correlation between the lower thymidine uptake and the reduced number of mitotic figures in the liver and cerebellum. Reduced thymidine uptake was found to occur between day 5 and day 6 of postnatal age; it persisted as long as there was a sufficient amount of DNA synthesis (or cell division). It was shown that there was a faster breakdown of DNA synthesized in the mutants, but that this faster breakdown was an irregular event, occurring in association with the late stage of the affliction. It was pointed out that the faster breakdown of DNA was not the basis for the neurological disorders. A lower uridine and leucine uptake was occasionally observed in the mutant organs at the late stage of affliction. This was pointed out as being associated with the atrophy of the organs. The level of RNA and protein showed a similar tendency. It was shown that a developmental lag in the total nitrogen level of the mutants was not a consistent phenomenon, and that this lag was independent from the behavioral abnormalities. It was shown that the metabolism of RNA, protein, and esterase in the mutant organs was relatively normal during the early stage of affliction in spite of the drastic reduction in the rate of DNA synthesis and cell division.