Decrease In Thymidine Uptake Research Articles

Selective Inhibition of HDAC1 and HDAC3 Inhibits Survival of Diffuse Large Cell Lymphoma Via Epigenetic Modulation of Death Associated Protein (DAPK)

Histone deacetylases (HDACs) are key players of epigenetic regulation of gene expression and are therefore attractive targets for cancer therapy. Many epigenetic drugs including HDAC inhibitors (HDACi) have shown anti-tumor activity in several types of cancers. Recently SAHA and belinostat Pan-HDACi have been FDA-approved for the treatment of T cell lymphoma. However, the capacity of Pan-HDACi for distinguishing different classes and isoforms of HDACs is limited, and may produce profound side effects. Therefore, it is important to identify cancer-relevant HDACs to promote selective HDAC inhibition for therapy in other malignancies such as diffuse large B cell lymphoma (DLBCL), the most aggressive non-Hodgkin lymphoma. Recent studies have shown that HDAC1 (HDACs class I) are commonly overexpressed in multiple cancer types and contribute to poor overall survival. However, the role of HDAC3 is not well known. The goal of this study was to determine the mechanistic role of HDAC3 and HDAC1 in DLBCL.Immunohistochemistry performed on DLBCL tumors (n=29) demonstrate 42% positivity for HDAC3 and 100% positivity for HDAC1. Ectopically expressed HDAC3 led to increased thymidine uptake, whereas HDAC3 knockdown using siRNA led to a significant decrease in thymidine uptake (p<0.001) suggesting that HDAC3 could be necessary for DLBCL cell growth. Moreover, inhibition of HDAC3 using a selective pharmacological inhibitor RGFP966 suppressed cell proliferation in a dose dependent manner in several DLBCL cell lines (SUDHL6, OCILY1, OCILy10 and OCILy3). However, the effect of RGFP966 on cell survival as determined by annexin V/ propidium iodide was underwhelming. We sought to target both HDAC1 and HDAC3 using the pharmacological inhibitor RG283 and assessed biological effect of dual inhibition. Interestingly, dual inhibition of HDAC1 and HDAC3 with RG2833 significantly suppressed both cell proliferation (<0.0001) and survival (p<0.001) in most of the DLBCL cell lines tested. As expected, treatment with RG2833 increased acetylation of H3K9, H3K14, H3K27 and H4K5 histone marks and led to P21 upregulation in DLBCL cells. Interestingly, exogenously overexpressed HDAC3 but not HDAC1 increased sensitivity of DLBCL cells to Pan-HDAC inhibitors vorinostat and belinostat, which suggest that endogenous HDAC3 in DLBCL cells may exist in a protein complex that may evade targeting by Pan-HDACi. In order to identify a potential mechanism of action for dual HDAC3 and HDAC1 inhibition, we have focused on tumor suppressor gene DAPK (Death associated protein), which has been previously shown to be silenced in DLBCL patients via DNA methylation. DAPK is a serine/threonine kinase and possesses pro-apoptotic and tumor-suppressive functions. We have confirmed by RT-PCR that DAPK was suppressed/silenced in DLBCL cell lines (n=8) as compared to CD19+ (n=3) normal B cells. HDAC inhibition alone or in combination with DNA methyltransferase inhibitors (DNMTi) can induce apoptosis by modulating expression of tumor suppressor genes. We next asked whether HDAC3 and HDAC1 dual inhibition through RG2833 alone or its combination with DNMTi, azacytidine (AZA) will be able to restore expression of DAPK in DLBCL cells. Quantitative RT-PCR analysis in OCILy3 and OCILY10 cells demonstrated that treatment with RG2833 alone modestly increased DAPK expression; however, co-treatment with RG283 and AZA led to marked increase in DAPK expression. As expected, the combination of RG2833 and Aza had significantly greater pro-apoptotic effect in DLBCL cells than either drug alone suggesting that DAPK expression may be an important determinant of response to HDAC1/HDAC3 dual inhibitor in DLBCL cells.In summary, our study provides first evidence of antitumor activity of dual HDAC3 and HDAC1 targeted agent therapy in DLBCL patients. Moreover, we have identified epigenetic modulation of DAPK as potential modulator of cellular response to HDAC1/HDAC3 dual inhibition. This study suggests for further clinical trials and/or development of selective inhibitors of HDAC3 or HDAC1/HDAC3 in DLBCL patients. DisclosuresNo relevant conflicts of interest to declare.

Decrease in Dipeptidyl Peptidase IV Activity is Linked to the Efficacy of Differentiating Compounds in Follicular Thyroid Carcinoma Cell Lines

Metastasized differentiated thyroid carcinoma is treated by surgery followed by radioiodine remnant ablation. The application of differentiating agents is a possibility of increasing the efficacy of radioiodine therapy. We evaluated DPP IV and aminopeptidase N, both linked to malignancy in thyroid carcinoma, and dipeptidyl peptidase II activities in human follicular thyroid carcinoma cell lines upon treatment with retinol, apicidine, and lovastatin as differentiating agents. Decrease of dipeptidyl peptidase IV (DPP IV) activity may play a role in the differentiating action. In the human cancer cell lines FTC 138 and 238, high DPP IV and low aminopeptidase N activities were recorded. Retinol treatment induced increases in thyroid-specific protein expression [thyroglobulin and sodium-iodide symporter (NIS)], increase in iodide uptake, and decrease in thymidine uptake accompanied by decrease in DPP IV activity. Decreases in DPP IV activities were also seen upon apicidine and lovastatin treatment, which also increased differentiation of the transformed thyrocytes. Our results demonstrate a link between decrease in DPP IV activity and increase in iodide uptake upon stimulation with differentiating agents.

The IGF-I/IGFBP system in congenital partial lipodystrophy.

Insulin and IGF-I interact at many levels. Little is known about the insulin-like growth factor-I/insulin-like growth factor binding proteins (IGF-I/IGFBP) system in congenital partial lipodystrophy, a syndrome characterized by insulin resistance, hyperinsulinaemia and absence of truncal and limb fat. Some cases have acromegaloid features with thick skin and large hands and feet in association with normal levels of circulating growth hormone. In four females known with congenital partial lipodystrophy, hyperinsulinaemia with acromegaloid features, the number and affinity of the IGF-I receptors on peripheral blood mononuclear cells (PBMCs), and the concentration of circulating insulin, total and free IGF-I, IGFBP-1 and IGFBP-3 levels were measured in the fasting and the fed state. Cultures of PBMCs of the patients with lipodystrophy were also used to study the effect of IGF-I stimulation on thymidine uptake in vitro. In the subjects with lipodystrophy the affinity and the number of the IGF-I receptors on peripheral mononuclear cells (PBMCs) and erythrocytes did not differ significantly from controls in the fasting state. Insulin levels were significantly higher in subjects with lipodystrophy both in the fasting as well in the fed state. Total IGF-I, free IGF-I and IGFBP-3 levels did not differ but serum IGFBP-1 levels were lower in lipodystrophy subjects than in healthy controls. The free IGF-I/IGFBP-1 ratio was increased in lipodystrophy subjects both in the fasting and the fed states. The effects of IGF-I stimulation on thymidine uptake by PBMCs of lipodystrophy subjects in the absence of IGFBP-1 were not different from healthy controls cultures in vitro. When a combination of IGFBP-1 (in a concentration comparable to the fasting serum IGFBP-1 levels in lipodystrophy patients found in our study) and IGF-I was added to PBMC cultures from lipodystrophy patients no decrease in thymidine uptake by PBMCs was found. In the four subjects with lipodystrophy hyperinsulinaemia, lowered free IGF-I and IGFBP-1 levels, but increased free IGF-I/IGBP-1 ratios were observed. Low IGFBP-1 concentrations in culture media did not reduce the stimulating IGF-I effect on thymidine uptake by PBMCs from lipodystrophy patients. Our data suggest that the observed increased IGF-I/IGFBP-1 ratio in lipodystrophy patients contributes to an unopposed biological effect of IGF-I on IGF-I receptors, thereby inducing the development of acromegaloid features, acanthosis nigricans and polycystic ovaries in some patients with congenital partial lipodystrophy.

Paradoxical elevation of Ki-67 labeling with protein kinase inhibition in malignant gliomas

The monoclonal antibody Ki-67 recognizes a nuclear antigen expressed in the G1, S, G2, and M phase of the cell cycle and has been used extensively as an indicator of cellular proliferation in malignant gliomas, both in the laboratory and clinically. Recently, protein kinase C (PKC) inhibitors have been demonstrated to inhibit malignant glioma growth both in in vitro and in vivo. This study was undertaken to determine whether Ki-67 could function as an indicator of cellular proliferation rate after PKC inhibition in gliomas and to explore cell cycle specificity of such inhibition. Both established and low-passage malignant glioma cell lines have previously been shown to be sensitive to growth inhibition by the PKC inhibitors staurosporine and tamoxifen in vitro (IC50 in the nanomolar and micromolar ranges, respectively), as measured by cell numbers, [3H]thymidine uptake, and flow-cytometric DNA analysis. However, in the same cells that are inhibited by staurosporine and tamoxifen on these assays, and on the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) assay in the present study, the Ki-67 labeling index paradoxically increased in a dose-related manner with the same treatments, as measured by immunohistochemistry and confirmed by flow cytometry. For example, in established line U-87, a 20.5% decrease in thymidine uptake and a 28.5% decrease in absorbance on the MTT assay produced by tamoxifen at 1 microM was associated with an increase in Ki-67 labeling from 42% to 62%; staurosporine, which produces a 78.8% decrease in thymidine uptake in cell line A-172 at 10 nM, produced an increase in Ki-67 labeling from 19% to 32%. In this regard, Ki-67 labeling of glioblastoma tissue from a patient treated with high-dose tamoxifen yielded results within the range of 10% to 15% (consistent with values seen in untreated glioblastoma), despite tumor regression with treatment. The authors' interpretation of these results is that these PKC inhibitors are halting the cell cycle in the G1 phase or the G1-S transition (beyond G0 but before S-phase), resulting in a paradoxical increase in labeling while arresting growth. Two important implications from these observations are that Ki-67 is not a reliable indicator of cellular proliferation after treatment with PKC inhibitors and that these inhibitors used at the doses given above halt cell growth in a phase-specific manner.

Ras Proteins Regulate Multiple Mitogenic Pathways in A10 Vascular Smooth Muscle Cells

We examined the role of ras proteins in mitogenesis and proliferation of A10 smooth muscle cells. An assay was developed in which a dominant negative ras gene could be transiently expressed in vascular smooth muscle cells. As opposed to cells transfected with a control plasmid, those transfected with a dominant negative ras expression plasmid exhibited a decrease in thymidine uptake in response to serum, platelet-derived growth factor, epidermal growth factor, fibroblast growth factor and thrombin stimulation. In addition, expression of dominant negative ras blocked smooth muscle cell proliferation as assessed by the number of surviving colonies after transfection and neomycin selection. Our data is the first to show that ras is essential for smooth muscle cell proliferation and is a mediator in multiple smooth muscle cell mitogenic signalling pathways.

Transforming Growth Factor-β1 Differentially Regulates Proliferation, Morphology, and Extracellular Matrix Expression by Three Neural Crest-Derived Neuroblastoma Cell Lines

We reported previously (S. L. Rogers, P. J. Gegick, S. M. Alexander, and P. G. McGuire, Dev. Biol. 151, 191-203, 1992) that transforming growth factor-β1 (TGFβ1) inhibited proliferation, up-regulated fibronectin synthesis, and suppressed melanogenesis in a population of quail neural crest cells in vitro. Here, we report that cell lines derived from the parent SK-N-SH neuroblastoma line (R. A. Ross, B. A. Spengler, and J. L. Biedler, J. Natl. Cancer Inst. 71, 741-747, 1983) respond differentially to TGFβ1, and their responses provide further insights into the actions of this growth factor on neural crest subpopulations. The SH-EP cell line exhibits primarily nonneuronal traits and responded to TGFβ1 with increased thymidine uptake after 6 days of culture, increased expression of fibronectin mRNA and protein, and decreased laminin synthesis. Many SH-EP cells also acquired a dramatically elongated morphology, reminiscent of Schwann cells in culture. Thymidine uptake by the neuronal SY5Y cell line was not substantially altered. Neither fibronectin mRNA nor protein was detectable in either TGFβ1-treated or untreated cultures, although laminin synthesis was upregulated by the growth factor. In TGFβ1-treated cultures of the intermediate SH-IN cell line, which has been reported to display both neuronal and nonneuronal characteristics, there was marked flattening of many cells, a steady decrease in thymidine uptake, and increased expression of both fibronectin and laminin. The observed responses of SH-IN cells mimic those observed in primary neural crest cultures and appear to represent similar differentiation toward a mesenchymal phenotype. These results substantiate the idea that closely related but diverging neural crest-derived cell types respond selectively to TGFβ1 and demonstrate that these SK-N-SH-derived cell lines will be useful in experimental approaches that will allow us to infer mechanisms underlying regulation of neural crest differentiation.

Fibroblast response to metallic debris in vitro. Enzyme induction cell proliferation, and toxicity.

Bovine synovial fibroblasts in primary monolayer culture were exposed to particulate metallic debris. The effects of the metallic particles on the synthesis and secretion of proteolytic enzymes and on cell proliferation and viability were examined. Uniform suspensions of titanium, titanium-aluminum, cobalt, and chromium particles, ranging in size from approximately 0.1 to ten micrometers (average, one to three micrometers), were prepared; the particle concentrations (the volume of particles divided by the total volume of the suspension) ranged from 0.0005 to 5 per cent. Aliquots of the particle suspensions were added to the synovial fibroblast cultures. The final particle concentrations in the media ranged from 0.0000083 to 0.83 per cent. After seventy-two hours of exposure, each medium was harvested and was assayed for proteolytic and collagenolytic activity and for hexosaminidase levels. Neutral metalloproteases, quantified by collagenolytic and caseinolytic (proteolytic) activity, represent enzymes, secreted by cells, that are capable of degrading extracellular matrix. Hexosaminidase is a marker for lysosomal enzyme activity that can include more than thirty enzymes, such as proteases, lipases, nucleases, and phosphatases. Cell proliferation was quantified by uptake of 3H-thymidine. Cell morphology was examined by scanning electron microscopy. Titanium, titanium-aluminum, and chromium significantly stimulated 3H-thymidine uptake at low particle concentrations (p < 0.01, p < 0.002, and p < 0.002, respectively). Exposure to cobalt, even at the lowest particle concentration, resulted in a significant decrease in thymidine uptake (p = 0.027). At the highest particle concentrations, all particles were toxic, as evidenced by the absence of thymidine uptake. At high particle concentrations, all of the metals caused a decrease in caseinolytic (proteolytic) and collagenolytic activity in the culture media. Titanium elevated the lysosomal enzyme marker, hexosaminidase, except at high concentrations. Chromium and titanium-aluminum had no significant effect on hexosaminidase at any particle concentration, while cobalt decreased all enzyme markers at mid-particle to high-particle concentrations. Scanning electron microscopy demonstrated that the morphological response of fibroblasts to titanium included membrane-ruffling and extension of filopodia, typical of active fibroblasts. In contrast, exposure to cobalt at the same concentration resulted in cell crenation, indicative of cell death.

Effect of pyridoxine on rifampicin toxicity.

The effects of pyridoxine (PN) on rifampicin (RMF) toxicity were investigated by both in vivo and in vitro methods. RMF (30 mg/kg) was injected intraperitoneally and PN(150 mg/kg) was administered orally to rats for 10 consecutive days. After treatment, clinical chemistry and hematologic profiles were measured. RMF and PN plus RMF did not show any adverse effects at this in vivo experimental condition. Thymidine incorporations of mice bone marrow cells were examined in vitro. RMF showed a decrease in thymidine uptake in a dose-dependent manner, but PN showed a reversal of the thymidine uptake suppression caused by RMF (p < 0.01). On the other hand, PN showed a decrease in thymidine uptake at a high concentration level.

Bi-functional action of transforming growth factor-beta on DNA synthesis in early passage human fetal fibroblasts.

We investigated the influence of transforming growth factor-beta (TGF-beta) on DNA synthesis in human fetal fibroblasts, as measured by the incorporation of [3H]thymidine and cell replication. In serum-free medium, without additional peptide growth factors, TGF-beta had no action on thymidine incorporation. However, in the presence of 0.1% v/v fetal calf serum, TGF-beta exhibited a bi-functional action on the cells. A dose-dependent stimulation of [3H]thymidine incorporation, and an increase in cell number, occurred with fibroblasts established from fetuses under 50 g body weight, with a maximum stimulation seen at 1.25 ng/ml. For fibroblasts from fetuses of 100 g or greater body weight, TGF-beta caused a dose-related decrease in thymidine uptake with a maximal inhibition at 2.5 ng/ml, and a small decrease in cell number. When DNA synthesis was stimulated by the addition of somatomedin-C/insulin-like growth factor I, epidermal growth factor, or platelet-derived growth factor, their actions were potentiated by the presence of TGF-beta on cells derived from fetuses under 50 g body weight, but inhibited on cells obtained from the larger fetuses weighing more than 100 g. Similar results were found for changes in cell number in response to TGF-beta when stimulated by SM-C/IGF I. The ability of TGF-beta to modulate [3H] thymidine incorporation did not involve a change in the time required for growth-restricted cells to enter the S phase of the replication cycle. These data suggest that TGF-beta may exert either a growth-promoting or growth-inhibiting action on human fetal connective tissues in the presence of other peptide growth factors, which is dependent on fetal age and development.

Mitogenic Activity of a Water-Soluble Adjuvant (Bu-WSA) Obtained from Bacterionema matruchotii: IV. Synergistic Effects of Bu-WSA on Concanavalin A-Induced Proliferative Response of Human Peripheral Blood Lymphocytes.

Butanol-extracted water-soluble adjuvant (Bu-WSA) obtained from Bacterionema matruchotii was cultured with peripheral blood mononuclear cells (PBM) in the presence of sub- and/or supra-optimal mitogenic concentrations of concanavalin A (Con A). The addition of Bu-WSA resulted in increased tritiated thymidine incorporation above that produced by Con A alone. Bu-WSA by itself is not mitogenic for PBM and in fact produced a decrease in thymidine uptake compared to the control. We investigated the response of subpopulation(s) of PBM to Bu-WSA, Con A and a mixture of Bu-WSA and Con A. Separation of PBM into purified T cells, B cells and macrophages showed that cell-cell cooperation of T cells with B cells or macrophages is necessary for the observed synergistic effect of Bu-WSA with Con A. A marked increase in thymidine incorporation by the mixture of T and B cell populations occurred, while only a small amount of thymidine was incorporated when the B cell population was absent. Mitomycin treatment revealed that the response could be ascribed to the T-cell response with a B-cell helper effect. Moreover, Con A and Bu-WSA appeared to act on the same T cell population. This model may provide unique information about the activation of human peripheral blood T cells compared with the activation of these cells by other mitogens.

EFFECT OF GROWTH HORMONE (GH) ON SOME ACTIVITIES OF LEUKOCYTES AND LYMPHOCYTES (LY) IN CHILDREN

We have shown in laboratory animals that GH stimulates several mitochondrial processes like protein synthesis, bulk-protein turnover, levels of eytochromes and enzymes, and respiratory activity. We have presently studied respiratory activity (RA) in white cells (WC) and thymidine uptake in LY of children before and after GH therapy. RA (ngm of O2/mg protein or/μg DNA) was measured in WC. prepared by layering whole blood on metrizoate-dextran medium, with a Clark-Oxygen electrode in Krebs-Ringer solution containing glucose. There is significant correlation (r =0.8) between percent increase in respiration due to GH therapy for 1 month and height Rain after 4-8 months of therapy in 16 children. This correlation may have significance in the early prediction of growth-promoting effect during therapy. LY, isolated by layering whole blood over Lymphoprep, showed an increase in WBC, LY count and rosette forming LY in 11-12 children but showed a decrease in 5-6 children. There is a significant decrease in thymidine uptake by unstimulated LY in all 12 children. Therefore, stimulation index (uptake with stimulation by phytohemagglutinim/uptake without stimulation) increased. These results suggest that GH therapy may reduce basal rate of DNA synthesis in LY.

Suppression of thymidine uptake of human lymphocytes by co-trimoxazole.

Co-trimoxazole (trimethoprim-sulphamethoxazole) causes a decrease in the uptake of labelled thymidine in lymphocytes cultured in the presence of phytohaemagglutinin. This phenomenon was observed in 60% of 25 subjects. In cultures affected by the drug the mean suppression was 84%. A small decrease in thymidine uptake was noted with trimethoprim and sulphamethoxazole separately, but the effect was much more pronounced with the combination of the two drugs. The mechanism responsible for this phenomenon is discussed. The action is probably not due to the ability of these drugs to interfere with folic acid metabolism and it is likely that there is no direct effect on DNA synthesis.The suppression of thymidine uptake by lymphocytes in vitro in the presence of co-trimoxazole may not have any obvious clinical significance. However, in view of a report of an immunosuppressive action of trimethoprim in mice, it is possible that the leucopenia observed in some patients treated with this drug may have been caused by a similar mechanism.These experiments show that lymphocytes in vitro are suppressed by co-trimoxazole in concentrations comparable to, or smaller than, those found in vivo under normal therapeutic conditions. They are therefore likely to be clinically relevant.

Differential effect of medium change in lymphocyte cultures stimulated by PPD tuberculin or PHA

The aim of this work was to study whether soluble factors produced in stimulated lymphocyte cultures play any role in the events in the cultures where they have been produced. When the whole culture medium in PPD-stimulated lymphocyte cultures was removed, either daily or on day 3, and replaced by fresh medium with the same antigen concentration, a marked decrease in thymidine uptake resulted. The same operation in PHA-stimulated cultures resulted in an increased thymidine uptake. Suitable controls showed that these effects were not due to artifacts, caused by manipulation of the cultures. If the old medium was replaced by medium from other stimulated lymphocyte cultures, the response was restored to normal levels. Medium from unstimulated cultures had a slight effect, which was considerably increased by addition of PPD. The findings may be related to unspecific conditioning factors, but possibly also to specifically lymphocyte-activating material.

Effects of mitomycin C on the infection of cells by polyoma virus and its DNA

Treatment of mouse embryo cell cultures with mitomycin C, sufficient to inhibit cell multiplication completely, led only to a partial decrease in uptake of thymidine. Such treatment of tissue culture cells before or after infection with polyoma virus or its infectious DNA virtually abolished production of virus or hemagglutinating viral antigen. The antibiotic, before or after chemical reduction, exerted no direct inhibitory effect on the infectious agents in vitro. However, there was a direct inhibitory effect on the virus and its isolated DNA when these were incubated with mitomycin in presence of the reducing agent dithionite. It appears, in confirmation of the studies of others, that mitomycin is converted by reduction to an extremely active form by the cellular enzyme system and that it is this form which interacts with both the viral or host cell genomes. Prereduced mitomycin appears to be rapidly inactivated by the aqueous media employed. Evidence is presented that cellular production of polyoma virus requires the integrity of both viral and host cell genomes.