14C-thymidine Incorporation Research Articles

In vivo tracking of 14C thymidine labeled mesenchymal stem cells using ultra-sensitive accelerator mass spectrometry

Despite the tremendous advancements made in cell tracking, in vivo imaging and volumetric analysis, it remains difficult to accurately quantify the number of infused cells following stem cell therapy, especially at the single cell level, mainly due to the sensitivity of cells. In this study, we demonstrate the utility of both liquid scintillator counter (LSC) and accelerator mass spectrometry (AMS) in investigating the distribution and quantification of radioisotope labeled adipocyte derived mesenchymal stem cells (AD-MSCs) at the single cell level after intravenous (IV) transplantation. We first show the incorporation of 14C-thymidine (5 nCi/ml, 24.2 ng/ml) into AD-MSCs without affecting key biological characteristics. These cells were then utilized to track and quantify the distribution of AD-MSCs delivered through the tail vein by AMS, revealing the number of AD-MSCs existing within different organs per mg and per organ at different time points. Notably, the results show that this highly sensitive approach can quantify one cell per mg which effectively means that AD-MSCs can be detected in various tissues at the single cell level. While the significance of these cells is yet to be elucidated, we show that it is possible to accurately depict the pattern of distribution and quantify AD-MSCs in living tissue. This approach can serve to incrementally build profiles of biodistribution for stem cells such as MSCs which is essential for both research and therapeutic purposes.

Synthesis and evaluation of 9-anilinothiazolo[5,4-b]quinoline derivatives as potential antitumorals

Five new 9-anilinothiazolo[5,4-b]quinoline derivatives (compounds 5, 7, 9, 10, 11) have been prepared. Some of the compounds were prepared by coupling properly substituted anilines to the novel compound 9-chloro-2-(methylthio)thiazolo[5,4-b]quinoline. Of these, compound 7 (9-anilino-2-[[2-( N,N-diethylamino)ethyl]amino]thiazolo[5,4-b]quinoline) showed the best cytotoxic activity in several cell lines. All compounds demonstrated DNA binding in nanomolar range. Compound 7 inhibited the 14C-thymidine incorporation into DNA. Results indicate that these derivatives deserve more considerations as potential antitumoral drugs.

PD0084430: a non-nucleoside inhibitor of human cytomegalovirus replication in vitro

Human cytomegalovirus (HCMV) is a major opportunistic pathogen in immunocompromised individuals. Current therapies target viral DNA replication and accumulate mutations that yield cross-resistance among the approved drugs. A novel, non-nucleoside inhibitor of HCMV replication, PD0084430, was identified in a screening assay using the HCMV β-galactosidase recombinant RC256. The EC 50 for PD0084430 by inhibition of β-galactosidase production is 1±0.7 μM. This antiviral activity was confirmed by yield reduction and plaque reduction assays using HCMV strain AD169. The TC 50 of PD0084430 as measured by 14C-thymidine incorporation is ∼30 μM and by XTT is ∼90 μM. The TC 50 for inhibition of cellular proliferation is ∼20 μM. Time of addition experiments displayed a similar drop in efficacy for both PD0084430 and GCV when added after the onset of viral DNA replication. The transcomplementation assay for viral DNA replication, using a transfected ori Lyt containing plasmid, confirmed that viral DNA synthesis was inhibited at the same concentrations that showed antiviral activity. Western blots showed no apparent block of immediate early or early gene expression. Two ganciclovir (GCV) resistant isolates of HCMV tested showed no cross-resistance to PD0084430. These data suggested a potentially promising novel compound that inhibited HCMV at or before viral DNA replication. However, in vivo testing in mice dosed either orally or intraperitoneally showed rapid glucuronidation on the –OH group. SAR studies on this backbone showed that the –OH group was essential for the antiviral activity in vitro.

Different impact of p53 and p21 on the radiation response of mouse tissues.

Mammalian tissues differ dramatically in their sensitivity to genotoxic stress, although the mechanisms determining these differences remain largely unknown. To analyse the role of p53 and p21 in determination of tissue specificity to DNA damage in vivo, we compared the effects of gamma radiation on DNA synthesis on whole-body sections of wild type, p53-deficient and p21-deficient mice. A dramatic reduction in 14C-thymidine incorporation after gamma irradiation was observed in the majority of rapidly proliferating tissues of wild type and p21-/- but not in p53-/- mice, confirming the key role of p53 in determination of tissue response to genotoxic stress in vivo and suggesting that p53-mediated inhibition of DNA synthesis does not depend on p21. Rapid radiation induced p53-dependent apoptosis was mapped to the areas of high levels of p53 mRNA in radiation sensitive tissues analysed (white pulp in the spleen and bases of crypts in small intestine), indicating that p53 regulation at the mRNA level is a determinant of cellular sensitivity to genotoxic stress. High p53 mRNA expression is inherited as a recessive trait in cell-cell hybrids suggesting the involvement of a negative control mechanism in the regulation of p53 gene expression.

A study of magnetic field effects on fibroblast cultures Part 1. The evaluation of the effects of static and extremely low frequency (ELF) magnetic fields on vital functions of fibroblasts

Cultured fibroblasts isolated from murine livers by tissue trypsinization were exposed to a static magnetic field (0.49 T) and an extremely low frequency (ELF) magnetic field (50 Hz, 0.020 T). The cultures were exposed to magnetic fields on four consecutive days for exposure times of 2, 4, 8, 16, 32 and 64 min. After exposure the following parameters of the fibroblast cultures were determined: the dynamics of culture growth, the protein content and 14C-thymidine incorporation. The cytometric parameters of the fibroblasts were also assessed. The ELF magnetic field compromised vital functions of the fibroblasts (inhibition of culture growth, decreased cellular protein, lowered cytometric parameters) and caused a slowdown in the rate of 14C-thymidine incorporation which indicates altered DNA synthesis. Ongoing experiments with a static magnetic field have shown no effect on the vital functions of fibroblasts.

Effects of amiloride on the induction of DNA synthesis and casein gene expression in rabbit mammary explants

Amiloride, an inhibitor of Na+/H+ exchange, was added at various concentrations to the culture medium of rabbit mammary explants. In the concentration range 100-250 microM, amiloride progessively inhibited 14C-thymidine incorporation induced by insulin, EGF or prolactin. Up to 250 microM, amiloride, which did not inhibit basal protein synthesis, was not cytotoxic, but it reduced basal DNA synthesis at the highest concentration. Addition of amiloride to the culture medium of mammary explants also strongly inhibited the induction of casein synthesis and casein mRNA accumulation by prolactin. The inhibition by amiloride is therefore not specific of the mitogenic action of prolactin since this drug also prevented its lactogenic action. The data reported here describe a new inhibitory action of amiloride on the transmission of the lactogenic signals.

Effect of temperature on alpha-amylase formation and DNA replication in Bacillus subtilis.

The effect of temperature on extracellular alpha-amylase synthesis and chromosomal and plasmid DNA replication in Bacillus subtilis A18 carrying plasmid pMI10 was studied. The specific growth rate mu increased with elevated temperature up to 42.5 degrees C, while the activities of alpha-amylase per population dry mass decreased. No obvious quantitative changes of 14C-thymidine incorporation per dry mass increase and no basic differences in plasmid copy number in the range of temperatures from 25 to 40 degrees C were found.

Polyamines and cell proliferation in the sea star Pycnopodia helianthoides

1. 1. Diamines (putrescine and cadaverine) and polyamines (spermidine and spermine) were extracted from tissues of the sea star Pycnopodia helianthoides, separated and quantitated using reversed-phase high-performance liquid chromatography (RP-HPLC). Simultaneous measurements of levels of protein and DNA and rates of incorporation of 14C-thymidine were carried out. 2. 2. The most abundant polyamine in tissues was spermidine (0.3873–2.5282 nmol/mg tissue) followed by spermine (0.103–1.5517 nmol/mg tissue), putrescine (0.2096–0.5322 nmol/mg tissue) and cadaverine (0.022–0.6064 nmol/mg tissue). 3. 3. An unknown molecule with derivatization and elution behaviour similar to that of polyamine standards was detected in all tissues. 4. 4. Protein levels ranged from 20.47 mg/g tissue in the body wall to 48.44 mg/g tissue in the pyloric caecum. 5. 5. DNA levels were lowest in the ovary (0.25 mg/g tissue) and highest in the testis (5.62 mg/g tissue). 6. 6. Incorporation of 14C-thymidine was highest in the testis. Testicular tissue had the highest spermidine/spermine ratio (5.4). A significant correlation between the spermidine/spermine ratio and 14C-thymidine incorporation (expressed either as DPM/g tissue or DPM/mg protein) suggests that polyamines are implicated in the regulation of cell proliferation in the sea star P. helianthoides.

Stimulation of bronchoalveolar lavage (BAL) and blood lymphocytes by Kveim antigen, tuberculin and concanavalin A in sarcoidosis

BAL and blood mononuclear cells and their reactivity to Kveim antigen, tuberculin and concanavalin A (Con A) were studied in nine patients with different clinical stages of sarcoidosis. After separation by plastic adherence, non-adherent cells (mainly lymphocytes) were admixed with 10% autologous adherent cells (monocytes/macrophages). After 3 and 6 days' culture with Kveim antigen (1, 10, 100 μg/ml), PPD tuberculin (2.5 μg/ml) and Con A (10, 20, 40 μg/ml) stimulation was measured as incorporation of 14C-thymidine into DNA. Except for occasional reactions the study did not show any unitary significant increase in lymphocyte response to the different concentrations of Kveim antigen in either BAL or blood. For Con A there was a weaker response by BAL-mononuclear cells with no difference between 3 and 6 days, compared with blood where there was an early peak. The lymphocyte reaction to PPD was weak with no difference between blood and BAL.

Estimating chemical developmental hazard in a chicken embryo limb bud micromass system.

A chicken limb bud micromass system measuring the production of cartilage proteoglycans and the incorporation of radiolabelled thymidine and leucine was used to calculate potential developmental hazard. This hazard index was based upon the 50% inhibitory concentration (IC50) for these parameters and enabled classification of substances according to their intrinsic ability to inhibit differentiation at concentrations which were not cytotoxic. All-trans-retinoic acid, a potent teratogen, inhibited cartilage proteoglycan synthesis at a concentration which was more than 100 times lower than those affecting the incorporation of 14C-thymidine and 14C-leucine. On the other hand, sodium valproate, salicylate and isoniazid inhibited cartilage proteoglycan synthesis only near concentrations which were also cytotoxic. The results from testing with chicken limb bud micromass compared favorably, both with respect to effective concentrations and ratios describing developmental hazard to those from rodent micromass systems.

Glucocorticoid receptor in multiple myeloma.

Glucocorticoid receptor levels of myeloma cells were quantitated in 7 patients with multiple myeloma. In 4 patients, glucocorticoid receptor levels were less than 10 fmol/10(6) cells. In 3 patients, receptor levels were from 17.6 to 21.4 fmol/10(6) cells. We further examined the correlation between glucocorticoid receptor levels and effect of dexamethasone on 14C-thymidine incorporation and cell viability using 2 myeloma cell lines, OPM-1 and OPM-2, established from a patient with multiple myeloma. Glucocorticoid receptor levels of OPM-1 and OPM-2 were 8.5 fmol/10(6) cells and 63.2 fmol/10(6) cells, respectively. The sensitivity of OPM-1 to dexamethasone in these studies was lower than that of OPM-2. These results suggest the possibility that the low level of glucocorticoid receptor in myeloma cells may be important for predicting a poor response to glucocorticoid therapy.

Effect of pyrimethamine and sulphadoxine on human lymphocyte proliferation

The in vitro effect of pyrimethamine (PYR) on human blood mononuclear cells stimulated with phytohaemagglutinin (PHA), pokeweed mitogen (PWM) and purified protein derivative of tuberculin (PPD) was studied by 14C-thymidine incorporation, by cell counting and by total DNA estimation. PYR in concentrations 10 times higher than serum values obtained in clinical practice inhibited lymphocyte proliferation irreversibly. PYR in concentrations corresponding to clinical practice quickly and irreversibly suppressed the proliferation of PWM-stimulated cells, and more slowly the proliferation of PPD-stimulated cells. The suppression of PHA-stimulated cells was reversed after one week. The increased 14C-thymidine incorporation observed in stimulated cells exposed to PYR in vitro in the early phase of proliferation did not reflect immunopotentiation but rather blocked endogenous thymidine synthesis. Sulphadoxine (SDX), added in vitro, had no effect on the lymphocytes, while SDX plus PYR had the same effect as PYR alone. Oral intake of SDX plus PYR (Fansidar) also blocked the thymidine synthesis of mitogen-stimulated lymphocytes. The possible consequences of the findings for the use of PYR in malaria prophylaxis and therapy are discussed.

Effect of oral proguanil on human lymphocyte proliferation.

In vitro studies have indicated that the antifolates pyrimethamine [4, 6] and cycloguanil (the active metabolite of proguanil) suppress the proliferation of stimulated human lymphocytes; proguanil has no effect [2]. During the early growth phase of the cells, 14C-thymidine (14C-TdR) incorporation is increased by pyrimethamine and cycloguanil, reflecting blockage of endogenous TdR synthesis [3]. Proguanil (Paludrine) is increasingly being used for malaria prophylaxis. It is considered the most innocuous of the antimalarials currently employed. Since nothing is known about the effect of oral proguanil on human lymphocytes, the present study was undertaken. Little information is available about the serum levels of proguanil and cycloguanil following ingestion of prophylactic doses [8]. Therefore, the serum concentrations of proguanil and cycloguanil were estimated, to allow comparison with previous in vitro studies [2].

Effect of chloroquine on human lymphocyte proliferation

The effect of chloroquine on human blood mononuclear cells was studied. High concentrations of chloroquine in vitro profoundly suppressed the proliferation of mitogen- and antigen-stimulated cells, as indicated by decreased 14C-thymidine incorporation. Lower concentrations of chloroquine increased the response to pokeweed mitogen. The response to concanavalin A and to various antigens was suppressed, especially the response to large particulate antigens. Oral intake of 300 mg of chloroquine base/week did not affect the lymphocyte proliferative responses. 600 mg of base/week decreased the response to large particulate antigens; the response to small antigens was not affected. The mode of action of chloroquine and the possible consequences of the findings for dosage of chloroquine when used for malaria prophylaxis is discussed.

Pyrimethamine-induced alterations in human lymphocytes in vitro. Mechanisms and reversal of the effect.

It has previously been shown that the antiprotozoal drug pyrimethamine (PYR) in concentrations corresponding to those obtained in clinical practice temporarily suppressed the proliferation of phytohaemagglutinin (PHA-) stimulated human lymphocytes in vitro; 10-fold higher concentrations permanently suppressed PHA-stimulated cells, as indicated by decreased numbers of cells and DNA synthesis. In the present study, it was found that the 3H-deoxyuridine incorporation in PHA-stimulated lymphocytes was suppressed by PYR, and that PYR caused defective deoxyuridine suppression of 14C-thymidine incorporation. The effects of PYR were completely corrected by low concentrations of folinic acid and high concentrations of folic acid, indicating that the basic mechanism of action of PYR is competitive blocking of dihydrofolate reductase. However, the effect of PYR was poorly corrected by exogenous thymidine; therefore, reduced thymidylate synthesis cannot be the sole consequence of PYR exposure. It is suggested that an additional folate-dependent factor plays an important role in the antimitotic activity of PYR on lymphocytes.

Immunodeficiency and prognosis in patients with non-Hodgkin's lymphomas.

Monocyte depleted blood lymphocyte subpopulations, their functions and relation to prognosis were studied in 68 untreated adult patients with non-Hodgkin's lymphomas classified according to the Kiel nomenclature. The median observation time was 48 months (range 41-60). The mean total blood lymphocyte and T (SRBC-rosetting) cell counts were significantly decreased as compared with age-matched controls (n = 57). Twenty-five per cent of the patients had a monoclonal blood B lymphocyte population. The spontaneous lymphocyte DNA synthesis, measured as incorporation of 14C-thymidine, was increased and the response to mitogen and antigen stimulation was decreased. Blood lymphocyte counts and functions before treatment were not related to the rates of remission or survival.

Effect of proguanil and cycloguanil on human lymphocytes in vitro.

The in vitro effect of proguanil and its active metabolite cycloguanil on proliferating human blood mononuclear cells was studied. Proguanil had no effect on 14C-thymidine incorporation or on the number of cells. Cycloguanil, in concentrations corresponding to the plasma levels found in clinical practice, blocked the endogenous synthesis of thymidine and decreased the number of mitogen- and antigen-stimulated cells. The effect on phytohaemagglutinin-stimulated cells was transient. Higher concentrations of cycloguanil, corresponding to intralymphocytic levels in clinical practice, permanently suppressed the growth of lymphocytes. The effect of cycloguanil could be reversed by low doses of folinic acid and high doses of folic acid.

Sensitivity of cultured human pancreatic carcinoma cells to dihydroxyanthracenedione.

We tested the effectiveness of dihydroxyanthracenedione (DHAD) on cell growth of two human pancreatic carcinoma cell lines MIA PaCa-2 and PANC-1. At the level of ID50, the drug was almost equally effective against both cell lines. When the time exposure of MIA PaCa-2 cells to the drug was increased from 1 h to continuous exposure for 5 days, the ID50 was decreased about three-fold only (1.4 X 10(-8)M and 4 X 10(-9)M respectively). At the level of ID50 also the difference between 6 h exposure and continuous exposure for 5 days was minimal. In equimolar concentrations and with 1 h exposure, DHAD was more effective against MIA PaCa-2 cells than other chemotherapeutic agents including adriamycin, mitomycin-C, 5-FU, vincristine, vindesine, vinblastine, VP-16-213, bleomycin, cis-platinum, asparaginase and acivicin. In concentrations of 5 X 10(-7)M, DHAD caused about 40% inhibition of 14C-thymidine incorporation of MIA PaCa-2 cells. Treatment of MIA PaCa-2 cells with the ID50 of DHAD for 1 h caused retardation of cellular traverse, with the major effect appearing to be in G2 + M phase of the cycle. From these data DHAD appears to be a potent drug against human pancreatic carcinoma in vitro.

Lymphocytotoxic serum factors and lymphocyte functions in untreated Hodgkin's disease.

The prevalence of complement dependent, cold-reactive lymphocytotoxic serum factors (LT) was studied in 80 untreated patients with Hodgkin's disease by a microcytotoxicity assay. Sera from 24 patients (30%) contained LT as judged from at least 50% lysis of lymphocytes from 16 to 23 randomly selected normal donors. The spontaneous incorporation of 14C-thymidine into blood lymphocytes from patients with LT was significantly higher than that of lymphocytes from LT-negative patients and from healthy controls. Total lymphocyte and T-cell counts, Concanavalin A, and pokeweed mitogen-induced lymphocyte DNA synthesis were lower in patients with LT. Lymphocytotoxic sera were more frequently encountered in patients with B symptoms, advanced disease, or nodular sclerosis/lymphocyte predominance histopathologic characteristics. LT were often found in patients with large and tumor-involved spleens. The ability of patient's serum to inhibit control lymphocyte response to Concanavalin A stimulation did not differ between LT-positive and LT-negative patients. We conclude that the presence of LT is associated with a quantitative and qualitative impairment of blood T-lymphocytes in untreated patients with Hodgkin's disease.

Biological activity of hormonally active and non-active androgen derivatives

Androgen derivatives appeared to have different biological activities in vivo and in vitro. Testosterone-17-isobutyrate given in three doses of 50 or 200 μ g increased significantly the weight of seminal vesicles and reduced thymus weight in castrated males, whereas testosterone-17-hemisuccinate, testosterone- D-β-glucoside and testosterone-3-( O-carboxymethyl)-oxime had no such effects. Similarly, ten 1-mg doses of testosterone-17-isobutyrate, unlike testosterone-17-hemisuccinate, resulted in a marked reduction of thymus weight in non-castrated males and in a significant inhibitory effect on the activity of spermatogenesis. On the other hand, all three androgen derivatives, which had appeared inactive in vivo, had similar effects in vitro (as had active testosterone) as demonstrated by inhibition of Concanavalin A-induced lymphocyte activation expressed by 14C thymidine incorporation and inhibition of cell agglutination. These results seem to suggest that as for the regulation of androgen-dependent organs and functions (such as the size of seminal vesicles, activity of spermatogenesis, thymus size), hormonally active androgens are also involved in certain immunosuppressive effects in vivo. On the other hand, in vitro immunological effects are produced by both hormonally active and non-active androgen derivatives as well as by other steroid hormones, the common denominator being the steroid structure.