Thymidine Uptake Assay Research Articles

Synthesis, structural properties and insulin-enhancing potential of bis(quercetinato)oxovanadium(IV) conjugate

An oxovanadium complex of quercetin ( 2), exhibits highly potent insulin-enhancing activity in streptozotocin-induced diabetic mice. It also mimics mitogenic potential of insulin as evaluated by [H 3]thymidine uptake assay making an effective, orally active insulin-enhancing agent for the treatment of both type 1 and type 2 diabetes without any noticeable toxic effects.

Concerted effect of transforming growth factor-beta, cyclin inhibitor p21, and c-myc on smooth muscle cell proliferation.

Increased aortic smooth muscle cell (SMC) proliferation is a key event in the pathogenesis of atherosclerosis. Transforming growth factor-beta (TGF-beta) is one of the potent inhibitors of SMC proliferation. The purpose of this study was 1) to explore the effect of TGF-beta inhibition on proliferation of SMC and expression of growth regulatory molecules like p21 and c-myc and 2) to determine whether restoration of cell cycle regulatory molecules normalizes the altered proliferation. To test the role of TGF-beta in SMC proliferation, using antisense plasmid DNA, we inhibited TGF-beta gene from aortic SMC, which resulted in a significant increase (P < 0.03) in proliferation (studied by quantifying new DNA synthesis with [(3)H]thymidine uptake assay). In TGF-beta-altered SMC (TASMC), the mRNA expression (studied by RT-PCR) of c-myc was increased whereas that of the cyclin inhibitor p21 was completely inhibited. Using p21 sense plasmid DNA, we transfected p21 gene in TASMC, which restored p21 mRNA and protein expression and decreased proliferation (P < 0.002) in TASMC. Similar treatment with c-myc antisense oligonucleotides significantly (P < 0.001) decreased the proliferation of TASMC. TASMC also exhibited alteration in morphological changes in SMC but returned to normal with treatment of p21 and TGF-beta sense plasmid DNA. Two-dimensional gel electrophoresis analysis of SMC and TASMC demonstrated differential expression of proteins relevant to cellular proliferation and atherosclerosis. This study uniquely analyzes the effect of TGF-beta at the molecular level on proliferation of SMC and on cell cycle regulatory molecules, implicating their potential role in the pathogenesis of atherosclerosis.

Abstracts

Pancreatic cancer, sometimes called a ‘silent killer’, is one of the most aggressive human malignancies, with a very poor prognosis. It is the fourth leading cause of cancer-related morbidity and mortality in the USA.A mouse peritoneal model was used to test the ability of unengineered rat umbilical cord matrix-derived stem cells (UCMSC) to control growth of pancreatic cancer. In vivo results were supported by various in vitro assays, such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), direct cell count, [3H]thymidine uptake and soft agar colony assays.Co-culture of rat UCMSC with PAN02 murine pancreatic carcinoma cells (UCMSC:PAN02, 1:6 and 1:3) caused G0/G1 arrest and significantly attenuated the proliferation of PAN02 tumor cells, as monitored by MTT assay, direct cell counts and [3H]thymidine uptake assay. Rat UCMSC also significantly reduced PAN02 colony size and number, as measured by soft agar colony assay. The in vivo mouse studies showed that rat UCMSC treatment significantly decreased the peritoneal PAN02 tumor burden 3 weeks after tumor transplantation and increased mouse survival time. Histologic study revealed that intraperitoneally administered rat UCMSC survived for at least 3 weeks, and the majority were found near or inside the tumor.These results indicate that naive rat UCMSC alone remarkably attenuate the growth of pancreatic carcinoma cells in vitro and in a mouse peritoneal model. This implies that UCMSC could be a potential tool for targeted cytotherapy for pancreatic cancer.

V- src oncogene-specific carboxy-terminal peptide is immunoprotective against Rous sarcoma growth in chickens with MHC class I allele B-F12

B 12 haplotype of the inbred chicken line CB ( B 12/ B 12) contains, like the bulk of chicken MHC( B) haplotypes, only a single dominantly expressed class I molecule (B-F). The peptide binding motifs for this major B-F12 molecule in chickens of Rous sarcoma regressor line CB ( B 12/ B 12) have been determined. Using stringent and relaxed motifs, several peptides were found in the v- src molecule of the PR-RSV-C, but most of these peptides are identical with that of endogenous c- src. Only the v- src C-tail peptide 517–524 (LPACVLEV) contains critical anchor amino acids (valine at positions 5 and 8) and shows a sequence different from the corresponding c- src peptide. This v- src C-tail peptide up-regulates expression of the B-F12 class I molecule on PBL, as assessed by FACS analysis, and stimulates T cell proliferation in a [ 3 H ]thymidine uptake assay. A protective effect of the immune response to LPACVLEV against RSV challenge was demonstrated in CB ( B 12/ B 12) chickens immunised with peptides encapsulated in liposomes.

Smad7 suppresses the inhibitory effect of TGF-beta2 on corneal endothelial cell proliferation and accelerates corneal endothelial wound closure in vitro.

The inhibitory activity of transforming growth factor-beta2 (TGF-beta2) on corneal endothelial cell proliferation is thought to be a cause of the limited regenerative capacity of corneal endothelial cells that may be related to impaired corneal transparency when many corneal endothelial cells are lost due to various stresses. We determined whether Smad7, an intracellular antagonist of TGF-beta signaling, regulated the inhibitory activity of TGF-beta2 or aqueous humor on corneal endothelial cell proliferation. The effect of Smad7 on TGF-beta2- or aqueous humor-mediated inhibition of corneal endothelial cell proliferation was evaluated using thymidine uptake assay with cultured rabbit corneal endothelial cells infected with adenovirus carrying Smad7. Expression of Smad or cell cycle-related proteins was detected by immunoblotting. In addition, a small scrape wound was made across a monolayer of Smad7-expressing cultured rabbit corneal endothelial cells to examine the effect of Smad7 on the wound-healing process in vitro. Overexpression of Smad7 abolished the inhibitory effect of TGF-beta2 or aqueous humor on the proliferation of cultured rabbit corneal endothelial cells associated with the inhibition of phosphorylation of Smad2 and downregulation of p27kip1. Smad7-overexpressing cultured rabbit corneal endothelial cells exhibited shorter wound closure time in the presence of aqueous humor than LacZ-expressing cells. Overexpression of Smad7 suppressed the inhibitory effect of TGF-beta2 or aqueous humor on corneal endothelial cell proliferation and accelerated corneal endothelial wound closure in vitro. Modification of Smad7 expression in corneal endothelial cells may thus have applicability in the treatment of wounded corneal endothelium.

Response to mitogens of chicken splenocytes determined by three methods of measurement

ABSTRACTThe aim of the present study was to examine whether the 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyl tetrazolium bromide (MTT) colorimetric assay can be applied to measurement of mitogen‐induced chicken splenocyte activation. Activation was also measured by a 3H‐thymidine uptake assay and a viable cell count assay. Optimal concentrations of mitogens and incubation periods required for maximal responses to mitogens differed between the MTT assay and the viable cell count and thymidine uptake assays. This probably reflects differences in the activities measured by the MTT assay which detects mitochondrial enzyme activity, and the thymidine uptake and viable cell count assays which detect cellular proliferation activity. The validity of the MTT assay was supported by the observation that the mitogen‐induced increase in succinate dehydrogenase activity paralleled the level of mitogen‐induced MTT formazan production. Mitogen concentrations inducing maximal formazan formation in chicken splenocytes were higher than those for chicken peripheral blood lymphocytes reported previously. Results of the present study indicate that mitogen‐induced chicken splenocyte activation could be measured by the MTT colorimetric assay, although mitogen concentrations and incubation periods required for maximal splenocyte activation differed between the MTT assay and the other two assays used in this study.

Beta-lapachone, a novel plant product, overcomes drug resistance in human multiple myeloma cells.

To evaluate the anti-tumor potential of beta-lapachone in multiple myeloma (MM) cell lines (U266, RPMI8226, and MM.1S); MM cell lines resistant to dexamethasone (MM.1R), melphalan (RPMI8226/LR5), doxorubicin (RPMI8226/DOX40), and mitoxantrone (RPMI8226/ MR20); and MM cells from patients (MM1-MM4). Cytotoxicity of beta-lapachone was assessed by MTT and [3H]-thymidine uptake assays. Apoptosis was analyzed using propidium iodide staining, DNA fragmentation, TUNEL assay, caspase-9 colorimetric assay, and immunoblotting for caspase-3, poly (ADP-ribose) polymerase (PARP), and caspase-8 cleavage products. Paracrine growth of MM cells was assessed by [3H]-thymidine uptake in cultures of bone marrow stromal cells (BMSCs) and MM cells. Interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) secretion in the culture supernatants was measured by specific enzyme-linked immunosorbent assays (ELISAs). beta-lapachone showed significant cytotoxicity in MM cells (IC(50): 4-8 microM). In contrast, normal peripheral blood mononuclear cells (PBMCs) and BMSCs from MM patients were relatively resistant (IC(50): 8-16 microM). IL-6 did not protect against beta-lapachone-induced apoptosis in MM.1S cells, and dexamethasone showed additive cytotoxicity. beta-lapachone also decreased binding of MM.1S cells to BMSCs; abrogated IL-6 and VEGF secretion triggered by adhesion of BMSCs to MM.1S cells; reduced proliferation of MM.1S cells adherent to BMSCs; and decreased intracellular adhesion molecule-1 (ICAM-1) expression on MM.1S cells. Furthermore, beta-lapachone induced typical PARP cleavage, increased caspase-9 proteolytic activity, and activation of caspase-3, without activation of caspase-8 in U266 cells. These studies provide a framework for clinical evaluation of beta-lapachone to improve the outcome for patients with MM.

Effect of pre-exposure to beta rays of tritium on some biochemical parameters measured in organs of rats subsequently irradiated with fast neutrons.

The experiment examined biological responses produced by combined sequential exposure to low-level tritium contamination, followed by challenging irradiation with fast neutrons. Modifications of endogenous antioxidant potential of different organs in rats were discussed in relation to tissue radiosensitivity. Rats pre-contaminated to 7 cGy and 35 cGy have been additionally irradiated to 1 Gy with fast neutrons. Lipid peroxide level was determined in liver, kidney, small intestine, spleen, bone marrow, and plasma. Reduced glutathione (GSH) level and glucose-6-phosphate dehydrogenase (G6PDH) activity were determined in erythrocytes. An in vitro thymidine uptake assay was performed in isolated bone marrow cells. The lipid peroxide level decreased significantly only in liver and kidney from rats pre-exposed to 35 cGy. For small intestine and spleen, tissues of comparatively higher radiosensitivity, no induced radioprotection was observed, as reflected in the homeostasis of the lipid peroxides. The same behavior was observed in bone marrow, the most radiosensitive tissue studied. However, the bone marrow thymidine-incorporation assay revealed a possible adaptive-type reaction in rats pre-exposed to 35 cGy. We conclude that for radiosensitive tissues pre-exposure to chronic low doses of low linear energy transfer (LET) irradiation has no protective effect on their antioxidant status, whereas a protective effect is observed in radioresistent tissues.

Partial characterization of endothelial FGF receptor functional domain by monoclonal antibody VBS-1.

Polypeptide growth factors mediate their cellular responses by binding to and activating specific cell surface receptors. Monoclonal antibody (MAb) VBS-1, produced against native fibroblast growth factor receptor-1 (FGFR-1), inhibited the binding of fibroblast growth factor-2 (FGF-2) to its receptor on coronary venular endothelial cells (CVECs) as determined by 125I-FGF-2 Scatchard analysis and [3H]thymidine uptake assays (ED50 = 80 ng/mL). Enzyme studies demonstrated that MAb VBS-1 binds to a protein epitope. Proteolytic mapping of the CVEC-FGFR established that a 52 kDa doublet contained the FGF binding site and the MAb VBS-1 antigenic epitope. N-glycanase digestion suggested the presence of a 50 kDa core protein for the CVEC-FGFR. Tunicamycin treatment resulted in the loss of expression of the core protein and the mature receptor, indicating the importance of CVEC-FGFR n-linked glycosylation. By Northern blot analysis, it was determined that CVECs express fgfr-1 and not fgfr-2. VBS-1 recognized FGFR-1 (140 kDa) and crossreacted weakly with FGFR-2 (135 kDa). Using a combination of affinity crosslinking, proteolytic mapping and Mab VBS-1 binding studies, we have located the FGF binding site near the NH2-terminal domain of the receptor close to the highly acidic box.

Antiproliferative and Apoptotic Effects of Iron Chelators on Human Cervical Carcinoma Cells

Objective. Cervical carcinoma is a human papillomavirus (HPV)-associated cancer for which treatment options still mainly rely on surgical procedures, with or without adjuvant radiotherapy and chemotherapy. As iron may participate in the pathogenesis of viral infections and cancer in several ways, the present study was designed to investigate the effect of iron chelation on HPV-16- and HPV-18-positive cervical carcinoma cell lines.Methods. Desferrioxamine and deferiprone, two chemically unrelated iron chelators, were used to investigate the effect of iron chelation on SiHa and HeLa cells. Proliferation was investigated by cells counts, by [3H]thymidine uptake assay, and by immunostaining with Ki-67 and proliferating cell nuclear antigen (PCNA). Apoptosis was determined by morphological analysis, by a TUNEL assay, and by flow cytometry detecting FITC-conjugated annexin-V.Results. Desferrioxamine and deferiprone induced a time- and dose-dependent inhibition of SiHa and HeLa cell growth. The inhibition of cell growth was associated with a decrease in the expression of both stable and total PCNA and Ki-67, a proliferation marker whose expression may predict survival in uterine cervical carcinoma. TUNEL assay, flow cytometry with annexin-V–fluorescein, and morphological analysis indicated that iron chelation also induced a time- and dose-dependent apoptosis of both cell lines. This apoptotic effect was prevented by the addition of exogenous iron.Conclusion. These results show that iron chelation inhibits the growth and induces the apoptosis of HPV-positive carcinoma cells. This suggests that iron chelators may represent a potential therapeutic approach for the management of cervical carcinoma.

Radiation sensitivity depends on OGG1 activity status in human leukemia cell lines

To assess the role of 8-oxoguanine glycosylase (OGG1) in the cell defense against radiation injury, the radiation-induced cytotoxicities were compared between the mutant type KG-1 featuring a loss of OGG1 activity due to a homozygous mutation of Arg 229 Gln, and the wild type U937. While the following three obvious toxicities were displayed in KG-1, they were observed only minimally in U937. These were: a dramatic arrest at the G2/M phase indicated by a marked increase in both the number of G2/M cells and the expression of cyclin B1, cdc2, and mitotic phosphoprotein monoclonal-2 (MPM-2)-reactive proteins; a severe apoptosis shown by a marked increase in the number of cells with hypo-diploid DNA and DNA fragmentation; and as a result, a severe inhibition of cell growth and proliferation measured by the MTT test and [ 3H]-thymidine uptake assay. As expected, KG-1 exhibited a significant increase in the 8-hydroxyguanine level in DNA whereas U937 did not. However, the level of irradiation-induced lipid peroxidation was almost the same in both cell lines. All of these symptoms shown by KG-1 were observed in Molt-4 and CEM-CM3, which were also found to feature low OGG1 activity. These findings suggest that OGG1 plays an important role in cell survival from radiation-induced damage and are also indicative of the capability of 8-hydroxyguanine in DNA to induce cellular toxicities.

2,3,7,8-Tetrachlorobenzo- p -dioxin Inhibits Proliferation of SK-N-SH Human Neuronal Cells Through Decreased Production of Reactive Oxygen Species

Oxidative stress has been known to be involved in the mechanism of toxic effects of various agents on many cellular systems. In this study we investigated the role of reactive oxygen species (ROS) in 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD)-induced neuronal cell toxicity using SK-N-SH human neuroblastoma cells. TCDD inhibited proliferation of the cells in a dose-dependent manner, which was revealed by MTT staining, counting of cells stained with trypan blue and [ 3 H]thymidine uptake assay. TCDD also suppressed the basal generation of ROS in a time- and concentration-dependent manner assessed by 2',7'-dichlorofluorescein fluorescence. In addition, TCDD induced a dose-dependent inhibition of lipid peroxidation, a biomarker of oxidative stress, whereas it significantly increased the level of glutathione (GSH), an intracellular free radical scavenger in the cells. Moreover, TCDD altered the activities of major antioxidant enzymes; increase in superoxide dismutase (SOD) and catalase, but decrease in glutathione peroxidase (GSH-Px) and glutathione reductase (GSH-Red). Pretreatment with l -buthionine- S , R -sulfoximine (BSO, 50 w M), an inhibitor of GSH synthesis, significantly prevented the TCDD-induced reduction in lipid peroxidation and cell proliferation. Interestingly, exogenous application of an oxidant, H 2 O 2 (50 w M) markedly restored the inhibited cell proliferation induced by TCDD. Taken together, these results suggest that alteration of cellular redox balance may mediate the TCDD-induced inhibition of proliferation in human neuronal cells.

The wound-healing effect of a glycoprotein fraction isolated from aloe vera.

Aloe vera has been used as a family medicine for promoting wound healing, but it is not known which component of the plant is effective for this purpose. To isolate and characterize the component effective in wound healing. Chromatography, electrophoresis and spectroscopic methods were used. The cell-proliferation activity of each component isolated was measured by a [3H]thymidine uptake assay. The cell-proliferation activity of the effective component was tested on a three-dimensional raft culture (cell culture technique by which artificial epidermis is made from keratinocytes). The effect of the active component on cell migration and wound healing was observed on a monolayer of human keratinocytes and in hairless mice. A glycoprotein fraction was isolated and named G1G1M1DI2. It showed a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis, with an apparent molecular weight of about 5.5 kDa. It exhibited significant [3H]thymidine uptake in squamous cell carcinoma cells. The effect of G1G1M1DI2 on cell migration was confirmed by accelerated wound healing on a monolayer of human keratinocytes. When this fraction was tested on a raft culture, it stimulated the formation of epidermal tissue. Furthermore, proliferation markers (epidermal growth factor receptor, fibronectin receptor, fibronectin, keratin 5/14 and keratin 1/10) were markedly expressed at the immunohistochemical level. The glycoprotein fraction enhanced wound healing in hairless mice by day 8 after injury, with significant cell proliferation. It is considered that this glycoprotein fraction is involved in the wound-healing effect of aloe vera via cell proliferation and migration.

In various tumour cell lines the peptide bradykinin B(2) receptor antagonist, Hoe 140 (Icatibant), may act as mitogenic agonist.

This study examined the mitogenic effects of bradykinin (BK, Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg), the peptide bradykinin B(2) receptor antagonist Hoe 140 (D-Arg(0)[Hyp(3)-Thi(6)-D-Tic(7)-Oic(8)]BK, and the orally active, nonpeptide B(2) receptor antagonist FR 173657 ((E)-3-(6-acetamido-3-pyridyl)-N-[N-2-4-dichloro-3-[(2-methyl-8-quino linyl) oxymethyl]phenyl]-N-methylaminocarbonyl-methyl]acrylamide) in three different human tumour cell lines: the small cell lung carcinoma (SCLC) cell line H-69, the breast carcinoma cell line EFM-192A, and the colon carcinoma cell line SW-480. In these cell lines activation of mitogen-activated protein kinase (MAPK) is involved in BK-induced stimulation of cell proliferation and may be mediated by both G(q) proteins (SW-480) and G(i) proteins (EFM-192A; H-69). In these cells BK as well as Hoe 140 increased the rate of DNA synthesis measured with the [(3)H]-thymidine uptake assay. Hoe 140 did neither antagonize nor potentiate the effect of BK. FR 173657 did not stimulate [(3)H]-thymidine incorporation but clearly antagonized the mitogenic effects of BK as well as Hoe 140. In H-69 cells, FR 173657 induced a decrease in the basal rate of DNA synthesis. In all three cell lines BK and Hoe 140 stimulated the activity of MAPK. Their effect on MAPK activity was completely abolished by FR 173657 which itself did not increase the activity of MAPK. In H-69 cells, the basal activity of MAPK was slightly inhibited by FR 173657. In the cell lines SW-480 and H-69 both BK and Hoe 140 but not FR 173657 stimulated phosphatidylinositol hydrolysis. In H-69 cells, FR 173657 decreased basal inositol phosphate formation. Our results show that in certain tumour cell lines the classical peptide B(2) receptor antagonist, Hoe 140, may act as mitogenic B(2) receptor agonist whereas the nonpeptide B(2) receptor antagonist, FR 173657, does not. In H-69 cells FR 173657 was found to exhibit properties of an inverse agonist.

Melatonin decreases cell proliferation and transformation in a melatonin receptor-dependent manner

There are conflicting claims for the role of melatonin in oncogenesis. In addition, the mechanism(s) underlying melatonin's effects in oncogenic processes is (are) unknown. In this study, the effects of melatonin exposure on cell proliferation and transformation were assessed in NIH3T3 cells transfected with either the human mt 1 (NIH-mt1) or MT 2 (NIH-MT2) melatonin receptors. The effects of melatonin exposure on proliferation was assessed by direct cell counts and [ 3H]thymidine uptake assays. The effect of chronic melatonin pretreatment on transformation was assessed by focus assays. In both NIH-mt1 and NIH-MT2 cells, melatonin pretreatment decreased cell proliferation and transformation. Control (NIH-neo) cells did not show this effect. However, as revealed by the [ 3H]thymidine uptake assays, an increase in DNA synthesis occurred in NIH-mt1 cells, whereas no increase occurred in the NIH-MT2 or NIH-neo cells. Upon examination of melatonin receptors, a decrease in the function of both mt 1 and MT 2 receptors occurred. These data suggest that perhaps an attenuation of receptor-mediated processes are involved in the anti-proliferative and anti-transformation capabilities of melatonin in NIH3T3 cells. In addition, based on the [ 3H]thymidine assays, receptor mediated signal transduction mechanisms may slow the growth of cells via actions on the cell cycle. The results from this study shed new insight on the putative mechanisms underlying melatonin's effects on cell proliferation and transformation and lends support for a protective role of melatonin in oncogenesis.

Interferon beta induces T-helper 2 immune deviation in MS.

To define the in vitro effects of interferon beta la (IFN-beta1a) on myelin basic protein (MBP)-reactive T cells and to determine its regulatory mechanism on cytokine networks in patients with MS. The proliferation and cytokine production of MBP-reactive T-cell clones were measured in thymidine uptake assays and ELISA respectively. The precursor frequency of MBP-reactive T cells was estimated in a microwell culture system. IFN-beta inhibited the proliferation of established MBP-reactive T-cell clones, which correlated with enhanced production of anti-inflammatory interleukin (IL)-4 and IL-10, and a decrease in tumor necrosis factor alpha (TNF-alpha) and IFN-gamma. When examined with peripheral blood mononuclear cells (PBMCs), IFN-beta was found to reduce the in vitro T-cell responses to MBP, as indicated by the significantly decreased frequency of MBP-reactive T cells. The decreased frequency of MBP-reactive T cells corresponded to an augmented production of IL-4 and IL-10. Although the level of TNF-alpha and IFN-gamma was generally unaltered or decreased, IFN-beta appeared to enhance the production of IFN-gamma in PBMCs derived from some individuals with MS. Interferon beta la (IFN-beta) suppresses myelin basic protein (MBP)-reactive T cells and induces immune deviation toward the production of T-helper 2 cytokines, which may contribute to its therapeutic benefit in MS. The study also suggests some heterogeneity in MBP-reactive T-cell responses to IFN-beta in different individuals with MS.

Purine Metabolites Suppress Proliferation of Human NK Cells Through a Lineage-Specific Purine Receptor

NK cell proliferation is suppressed in some patients with cancer by unknown mechanisms. Because purine metabolites released into the extracellular space during cell lysis may affect cell function, we hypothesized that these metabolites could serve as feedback regulators of NK cell proliferation. Sorted NK (CD56+/CD3-) cells were incubated with IL-2 (1000 U/ml) in a 4-day thymidine uptake assay with or without 10-10,000 microM of nucleotides. Adenine nucleotides inhibited NK cell proliferation, with ATP = ADP > 5'-adenylylimidodiphosphate > AMP = adenosine; ADP-ribose and nicotinamide adenine dinucleotide, but not nicotinamide or UTP, caused a dose-dependent suppression of thymidine uptake. A total of 100 microM ATP, a concentration that induced a maximal (80%) inhibition of thymidine uptake, did not inhibit cytotoxic activity against K562 targets. Because NK cells retained the ability to lyse K562 targets 4 days after exposure to 500 microM ATP or 1000 microM adenosine, inhibition of thymidine uptake was not due to cell death. Incubation of NK cells with dibutyryl cAMP and forskolin also suppressed thymidine uptake. Cholera toxin and pertussis toxin suppressed NK cell proliferation. Pertussis toxin did not block the adenine nucleotide effects. Further, ATP, but not adenosine or other nucleotides, markedly increased intracellular cAMP in a dose-dependent manner. The ATP-induced increase in cAMP was specific to cytolytic cells, because CD19+ B cells and CD4+ T cells did not increase their intracellular cAMP. These studies demonstrate that NK proliferation is regulated through purine receptors by adenine nucleotides, which may play a role in decreased NK cell activity. The response to adenine nucleotides is lineage-specific.

Transforming growth factor-beta1 up-regulates p15, p21 and p27 and blocks cell cycling in G1 in human prostate epithelium.

Transforming growth factor-beta1 (TGFbeta1) is inhibitory to most epithelia, but its role in the control of proliferation of prostatic epithelium is unclear. In some cells, TGFbeta1 inhibition is achieved by up-regulation of cyclin-dependent kinase (cdk) inhibitors including p15, p21 and p27. Our aims were to determine whether the effects of TGFbeta1 on human prostatic epithelial cell cycle kinetics were mediated by alterations in the levels of the cdk inhibitors p15, p16, p21 and p27 and hypo-phosphorylated retinoblastoma protein (Rb). Human prostatic epithelial cells in primary culture were grown in the presence of TGFbeta1 (0-10 ng/ml) for up to 4 days and proliferation assessed using a [3H]thymidine uptake assay. Levels of p15, p16, p21 and p27 were measured at both mRNA and protein level by means of a reverse transcriptase PCR-based assay and Western analysis. Rb and cdk2 levels were measured. Exogenous TGFbeta1 (0-5 ng/ml) inhibited proliferation. This was associated with blocking of the cell cycle at G1, and up to 4-fold increases in p15, p21 and p27 mRNA levels, but no change was observed in p16 mRNA levels; these changes were not blocked by cycloheximide. Increased levels of p15, p21 and p27 protein were also accompanied by increased levels of hypo-phosphorylated Rb and decreased cdk2 kinase activity. TGFbeta1 has mainly inhibitory effects on benign human prostatic epithelium, which are caused by up-regulation of cdk inhibitors, hypo-phosphorylation of Rb and delaying of the cell cycle in G1.

SUPERFICIAL BLADDER TUMORS AND INCREASED REACTIVITY AGAINST MYCOBACTERIAL ANTIGENS BEFORE BACILLUS CALMETTE-GUERIN THERAPY

Purpose The precise mechanism of action of bacillus Calmette-Guerin (BCG) in bladder cancer treatment remains poorly understood. Whether bladder tumor cells are destroyed by nonspecific mechanisms or targeted by specifically activated lymphocytes recognizing cognate antigens is unclear. To investigate a possible cross-reactivity between BCG and bladder cell tumors, we tested before BCG treatment the lymphoproliferation of peripheral blood lymphocytes against several mycobacterial antigens, including the secreted fibronectin binding antigen 85 complex from BCG (AG 85) in patients with superficial bladder tumors compared to control matched patients. Materials and Methods Using a whole blood assay, T cell response against purified protein derivative, BCG extract, whole BCG, purified AG 85, and the nonspecific mitogens pokeweed and phytohemagglutinin was investigated in 79 patients with superficial bladder tumors before BCG and in 39 control subjects without malignancy matched for age and sex. Neither group had a history of tuberculosis. Lymphoproliferation was measured with a tritiated thymidine uptake assay on day 7 of culture. Results Of the 79 patients with superficial transitional cell carcinoma, a significant lymphoproliferative response before BCG against PPD, BCG extract, whole BCG and AG 85 was observed in 65 (82.2%), 67 (84.81%), 30 (37.97%) and 49 (62.02%) patients, respectively. Of the 39 controls only 26 (64.1%), 23 (58.9%), 3 (7.7%) and 3 (7.7%) patients, respectively, had a significant lymphoproliferation against PPD, BCG extract, BCG and AG 85 (p >0.05, p = 0.004, p = 0.00001 and p = 0.00001, respectively). In terms of lymphoproliferative levels, patients with superficial transitional cell carcinoma also showed a significantly higher response against PPD (p = 0.000012), BCG extract (p = 0.000001), AG 85 (p = 0.000001), whole BCG (p = 0.00001) and pokeweed (p = 0.01) than controls but not against phytohemagglutinin. Conclusions Patients with superficial transitional cell carcinoma demonstrate an increased lymphoproliferation against mycobacterial antigens before BCG compared to control subjects. Although a nonspecific activation of the immune system cannot be excluded at this stage, our data may suggest the possible existence of bladder cancer antigens cross-reactive with mycobacterial antigens responsible for boosting precursor cells witnessing previous contacts with mycobacteria. The implication of these findings in the antitumoral mechanism of action of BCG are under investigation.

The inhibitory effect of ursodeoxycholic acid and pentoxifylline on platelet derived growth factor-stimulated proliferation is distinct from an effect by cyclic AMP

This study assessed the ability of ursodeoxycholic acid (UDCA) and one of its metabolites, tauroursodeoxycholic acid (TUDCA), to inhibit platelet derived growth factor (PDGF) stimulated fibroproliferation and compared these results to the effect of pentoxifylline and its metabolite-1 [1-(5-hydroxyhexyl)-3,7-dimethylxanthine] and assessed the potential role of cyclic AMP in this process. Fibroproliferative activity was measured by the tritiated thymidine uptake assay in human fibroblast cultures. All four compounds: pentoxifylline, metabolite-1, UDCA and TUDCA inhibited the fibroproliferative activity stimulated by PDGF (8 ng/ml). Incubation of fibroblasts with dibutyryl cyclic AMP reduced proliferation stimulated by PDGF suggesting that the PDGF stimulated proliferation was sensitive to inhibition by a membrane permeable analogue of cyclic AMP. Incubation of myofibroblasts with dibutyryl cyclic AMP significantly inhibited PDGF stimulated proliferation suggesting that cyclic AMP can regulate PDGF stimulated proliferation in the myofibroblast. To determine if the effect of pentoxifylline on fibroproliferation was mediated by cyclic AMP, we used dideoxyadenosine, a potent inhibitor of adenylyl cyclase. The effect of pentoxifylline on fibroproliferation was not prevented by dideoxyadenosine, which inhibits formation of cyclic AMP, thus suggesting that the inhibitory effect of pentoxifylline on PDGF-stimulated proliferation of fibroblasts was not mediated by cyclic AMP, arguing against a role for cyclic AMP in this process. Combinations of UDCA (250 μM) plus pentoxifylline (120 μM) or UDCA (250 μM) plus TUDCA (250 μM) inhibited fibroproliferative activity stimulated by PDGF to a greater extent than either drug alone. As UDCA has been reported to decrease cyclic AMP these results argue against a role for cyclic AMP in this process. Finally the results suggest that UDCA may inhibit PDGF-stimulated proliferation via an inhibition of C-kinase.