Inhibition Of Tritiated Thymidine Incorporation Research Articles

Therapeutic targeting of Src-kinase Lyn in myeloid leukemic cell growth.

Protein tyrosine kinases play a major role in promoting cell growth, and their activity in solid tumors is well established. Inhibitors of protein tyrosine kinases are now in advanced clinical trials for the treatment of breast and brain cancers. Because Src-related PTK have been shown to be activated in leukemic cell lines, we studied their activation in human myeloid leukemia. Blasts from the majority of patients with acute leukemia showed constitutive activity of the Src kinase Lyn. In contrast, no patient samples showed constitutive activation of Jak2. Genetic and pharmacologic targeting of Lyn was used to determine its contribution to leukemic cell growth. Antisense Lyn oligonucleotide treatment resulted in the inhibition of tritiated thymidine incorporation following GM-CSF stimulation of the factor-dependent line MO7e. The Src kinase inhibitor PD166285 inhibited the growth of human leukemic cell lines and leukemic blasts. When combined with doxorubicin, an additive effect on the inhibition of leukemic cell growth occurred. These studies demonstrate the importance of Src kinases in promoting leukemic cell growth and suggests that further development of agents which target Src kinases and their inclusion in multidrug regimens are warranted for novel therapies of myeloid leukemia.

Expression and Regulation of Transforming Growth Factor β1 mRNA and Protein in Rat Fetal Testisin Vitro

The expression and secretion of Transforming Growth Factor β1 (TGFβ1) by cultured testes of day 20.5 rat fetuses were investigated. The testes were found to express two TGFβ1 mRNA transcripts of 2.5 and 1.8 kb. By using mink lung epithelial cell bioassay based on the measurement of the inhibition of tritiated thymidine incorporation in response to TGFβ1 immunoreactive material, the fetal testes were shown to secrete TGFβ1 protein in organ culture. This secretion was positively regulated by dibutyryl cyclic AMP or by LH and FSH together, but not by LH alone and very slightly by FSH alone, which suggests interactions between Leydig and Sertoli cells for the control of TGFβ1 production. These regulations probably take place at a posttranscriptional step since no concomitant increase of TGFβ1 mRNA levels was observed. Such a positive regulation of TGFβ1 secretion by gonadotropins could be a characteristic of the rat fetal testis.

Influence of Uremic Middle Molecules on In Vitro Stimulated Lymphocytes and lnterleukin-2 Production

The influence of uremic middle molecules on in vitro lymphocyte blast transformation and interleukin-2 production was studied in 12 patients on long-term hemodialysis and 12 healthy control subjects with normal kidney function. Middle molecules inhibit phytohemoagglutinin induced lymphocyte proliferation in a concentration dependent fashion, achieving more than 50% inhibition of tritiated thymidine incorporation after 72 hrs in culture. In addition, there was less interleukin-2 production in the long-term hemodialysis patients studied compared with healthy control subjects.

Characterization of an antiproliferative surface-associated protein from Actinobacillus actinomycetemcomitans which can be neutralized by sera from a proportion of patients with localized juvenile periodontitis.

The gentle agitation of suspensions of Actinobacillus actinomycetemcomitans serotype a, b, or c in saline resulted in the release of a proteinaceous surface-associated material (SAM) which produced a dose-dependent inhibition of tritiated thymidine incorporation by the osteoblast-like cell line MG63 in culture. This cell line was sensitive to low concentrations of SAM (50% inhibitory concentration, 200 ng/ml for serotype c). Immunoglobulin G antibodies to constituents of the SAM were found in the blood of patients with localized juvenile periodontitis (LJP). Sera from 9 of 16 patients with LJP significantly neutralized the antiproliferative activity of the SAM, while sera from 15 controls, with no evidence of periodontal disease, were unable to neutralize this activity. Neutralization was not directly related to the patient's antibody titer to the whole SAM. Characterization of the antiproliferative activity in the SAM demonstrated that it was not cytotoxic and was heat and trypsin sensitive. The active component separated in a well-defined peak in anion-exchange high-performance liquid chromatography (HPLC) which, when further analyzed by size exclusion HPLC, revealed a single active peak, which had an apparent molecular mass of approximately 8 kDa. The lipopolysaccharide from A. actinomycetemcomitans was only weakly active. SAM from Porphyromonas gingivalis W50 and Eikenella corrodens NCTC 10596 did not exhibit any antiproliferative activity with this cell line, even at concentrations as high as 10 micrograms/ml. This study has shown that SAM from A. actinomycetemcomitans contains a potent antiproliferative protein whose activity can be neutralized by antibodies in the sera from some patients with LJP.

Generation of cytotoxic antibodies to the B16 murine melanoma using a formalinized vaccine.

The goal of our experiments was to determine the extent to which the humoral response to a melanoma vaccine elicits the production of cytotoxic antibodies in tumor-challenged mice. Mice were immunized with a vaccine produced from formalinized extracellular antigens (FECA) derived from B16 F10 melanomas. The production of antibodies that recognized the vaccine preparation was determined by ELISA, as was their cross-reactivity with the B700 melanoma antigen. The antibodies were shown to be anti-proliferative by inhibition of tritiated thymidine incorporation into the DNA of cultured target cells and cytotoxic by assays for complement-mediated and antibody-dependent cellular cytotoxicity. Flow cytometric analyses indicated that approximately 60% of the target cells specifically bound antibody from the immune sera. These results confirm that B700 is a significant antigenic component of the FECA vaccine, and provide encouragement for this approach to developing useful melanoma vaccines.

The effect of antibodies and immunotoxins reactive with HER-2/ neu on growth of ovarian and breast cancer cell lines

Because HER-2/neu is overexpressed in one third of breast and ovarian cancers, we examined the effect of unconjugated monoclonal antibodies (ID-5, PB-3, TA-1) and an immunotoxin (TA-1-ricin) reactive with this protooncogene on the growth of breast and ovarian cancer cell lines. The tritiated thymidine incorporation assay was used to examine the effect of unconjugated antibodies on proliferation. A limiting dilution clonogenic assay was used to assess the effect of immunotoxin on cellular cytotoxicity. Scatchard analysis revealed that OVCA 420, OVCA 429, OVCA 432, and OVCA 433 cells had approximately 10(4) HER-2/neu receptors per cell, whereas the SKOv3 and SKBr3 cell lines expressed 10(5) and 10(6) receptors per cell, respectively. Monoclonal antibody ID-5 caused significant inhibition of tritiated thymidine incorporation in SKBr3, SKOv3, and OVCA 420 cells (p < 0.002). The TA-1-rich immunotoxin significantly inhibited the clonogenic growth of only SKBr3 and SKOv3 cells. HER-2/neu may be a useful target for immunotherapy with unconjugated antibodies and immunotoxins in ovarian and breast cancers that overexpress this protooncogene.

Effects of suramin on in vitro growth of fresh human tumors.

Suramin is a polysulfonated urea recently tested in clinical trials as an anticancer agent. To define tumor types for further clinical testing of suramin, we assessed the in vitro activity of suramin against fresh human tumor specimens. Inhibition of tumor colony formation (human tumor clonogenic assay [HTCA] method) and inhibition of tritiated thymidine incorporation (TTI method) were used as indicators of drug sensitivity. With the use of the HTCA method, 80% or more of carcinomas of the colon, endometrium, kidney, lung (non-small-cell), and ovary as well as malignant melanoma and mesothelioma were sensitive to 200 micrograms/mL of suramin by continuous exposure. Suramin's antitumor activity was dose dependent, and it was less effective when tested at concentrations of 50 micrograms/mL or less. With the TTI method, the more slowly growing tumors (breast cancer, colon cancer, multiple myeloma, non-Hodgkin's lymphoma, prostate cancer, and sarcoma) appeared to be less sensitive to suramin. However, when the two assay methods were directly compared in melanoma and ovarian cancer specimens, individual tumors were generally more sensitive to suramin (greater inhibition relative to control) using the HTCA method, whereas the TTI method appeared to underestimate suramin's antitumor activity. There was no significant difference in the activity of suramin when tested in the presence of 10% versus 50% serum. These results provide an experimental basis for clinical evaluations of suramin therapy in patients with colon, endometrial, kidney, non-small-cell lung, and ovarian cancers as well as malignant melanoma and mesothelioma.

Uptake and cytotoxicity of novel nitroimidazole-polyamine conjugates in ehrlich ascites tumour cells

A number of tumour cells, including Ehrlich ascites tumour cells (EATC), possess a polyamine uptake system which selectively accumulates endogenous polyamines and structurally related compounds by an active energy dependent system(s). We suggest that it may be possible to utilize this uptake system to target certain cytotoxic agents to those tumour cells possessing this system. In an initial attempt to determine the feasibility of this suggestion, we have synthesized a series of 2- and 5-nitroimidazoles linked to polyamines and determined their ability to utilize the polyamine uptake system. Within the limited series of compounds synthesized, 2-nitroimidazole-polyamine conjugates were more potent inhibitors of spermidine uptake into EATC than the 5-nitroimidazole conjugates. It has been assumed partly based on the competitive nature of this inhibition, that the ability of these compounds to inhibit spermidine uptake is also a measure of their ability to be accumulated by EATC. A greater than 700-fold variation was observed in the ability of different analogues to inhibit spermidine uptake. The most potent inhibitors retained certain structural characteristics similar to those of spermidine. Those compounds linked to polyamines were much more potent inhibitors of polyamine uptake than the parent nitroimidazoles i.e. metronidazole and misonidazole. The toxicity of the parent compounds and their polyamine conjugates in control and polyamine-depleted EATC was assessed by measuring inhibition of tritiated thymidine incorporation. Polyamine depletion, by prior exposure to difluoromethylornithine, results in a compensatory increase in the uptake of polyamines and related structures which may result in an increase in toxicity. Whilst many of the novel conjugates showed only little or moderate toxicity to control cells, the toxicity of several of the conjugates but not the parent nitroimidazoles increased in the polyamine-depleted cells. A clear distinction was also observed between the ability to inhibit spermidine uptake (and hence affinity for the uptake system) and toxicity, e.g. compound 430, a dinitroimidazole-polyamine conjugate, was the best inhibitor of spermidine uptake studied but showed no toxicity. These results support the hypothesis that linking polyamines to nitroimidazoles facilitates the entry of the latter into cells, such as EATC, which possess the polyamine uptake system and may therefore have therapeutic application in the delivery of polyamine-linked cytotoxics to certain tumours.

A rapid aquatic toxicity assay utilizing labeled thymidine incorporation in sea urchin embryos

AbstractAquatic toxicity was evaluated in the sea urchin embryo (Arbacia punctulata) by the inhibition of tritiated thymidine incorporation after a brief exposure to toxic chemicals. In preliminary trials, using eight compounds of varying toxicities, results accurately predicted reduced survival and morphological delay after 48‐h exposures (r2 = 0.97). Excellent correlations also exist between these calculated median effective concentrations (EC50 values) and literature‐derived acute LC50 values for two other aquatic species. Arbacia is a useful surrogate species for assay: well‐studied, easily cultured and fertile virtually year round. The simplicity and speed of this test system lends itself to screening large numbers of compounds, mixtures or water samples.

Inhibition of T lymphocyte mitogenesis by 1,25-dihydroxyvitamin D3 (calcitriol).

Recent studies have suggested that vitamin D may have other important biologic activities in addition to its well-characterized role in the maintenance of calcium homeostasis. Discovery of cytosolic receptors for vitamin D in human peripheral blood monocytes and lectin-stimulated lymphocytes prompted us to study the effects of 1,25-dihydroxyvitamin D3 (calcitriol), the most biologically active metabolite of vitamin D, upon phytohemagglutinin (PHA)-induced lymphocyte blast transformation. We have found that calcitriol is a potent inhibitor of PHA-induced lymphocyte proliferation, achieving 70% inhibition of tritiated thymidine incorporation after 72 h in culture. Furthermore, calcitriol suppressed interleukin-2 (IL-2) production by PHA-stimulated peripheral blood mononuclear cells in a concentration-dependent fashion. Lastly, the suppressive effect of calcitriol on cellular proliferation was partially reversed by the addition of saturating amounts of purified IL-2. We conclude that calcitriol is a potent inhibitor of PHA-induced lymphocyte blast transformation and that this effect is mediated, in part, through suppression of IL-2 production. Thus, calcitriol appears to possess immunoregulatory properties that have been unappreciated heretofore.

Depigmenting Action of Hydroquinone Depends on Disruption of Fundamental Cell Processes

The effect of the skin-depigmenting agent hydroquinone (HQ) on 2 melanotic and 3 nonmelanotic cell lines was studied. Significant differences in its effect on DNA and RNA synthesis were observed between cell lines. HQ caused inhibition of cellular metabolism in all cells tested, but the dose that caused 50% inhibition of tritiated thymidine incorporation was approximately 30 times lower for melanotic cells. Tritiated uridine incorporation was found to be 85 times more sensitive to HQ in the melanotic cells. These results suggest that HQ exerts its depigmenting effect by selective action on melanocyte metabolism rather than a specific effect on melanin synthesis. Further, the effects of UV irradiation on this system were investigated and found to be negligible, in spite of the stimulation of in vivo melanin synthesis by UV radiation.

Inhibition of Tritiated Thymidine Incorporation in Cultured Cells by Rat Kidney Extract&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;

KCl extract from rat kidney, rat liver, and Morris hepatomas inhibited [3H]thymidine incorporation into cultured cells. Tissues came from male inbred BUF rats. The most pronounced inhibition was achieved with the kidney extract. Protein synthesis was not inhibited during a 24-hour exposure of the cells to the inhibitor. Incorporation of [3H]deoxycytidine was inhibited, as was cell growth, when the kidney KCl extract was present for several days. [3H]thymidine incorporation was inhibited almost immediately after the addition of the extract. The inhibition was reversible. Regular [3H]thymidine incorporation was restored 24 hours after removal of the inhibitor, which was neither arginase nor a thymidine-degrading enzyme. The inhibitor was stable to heat (80 degrees C for 10 min) and resistant to trypsin, pronase, DNase, and RNase. Exposure of the extract to proteolytic enzymes, hyaluronidase, and neuraminidase resulted in a loss of inhibitory activity only after extensive dialysis of the treated extract. The inhibitor appeared to be a mucoprotein in which the carbohydrate moiety may be responsible for the inhibition. The KCl extract also inhibited RNA synthesis and DNA synthesis by the de novo pathway. The inhibition of phosphorylation of thymidine, however, appeared to be the primary action of the inhibitor.

Glucocorticoid Receptor and Effector Mechanisms: A Comparison of the Corticosensitive Mouse with the Corticoresistant Guinea Pig*

In terms of steroid effector systems, one of the areas least explored is the often described species difference in sensitivity to administered glucocorticoids. We have compared a species reported to be corticosensitive (mouse) with a more corticoresistant species (guinea pig) in terms of 1) the affinity with which tritiated dexamethasone ([3H]DM) is bound in spleen cytoplasmic preparations from adrenalectomized animals, 2) the sensitivity to DM of phytohemagglutinin-treated spleen cells in vitro, measured by inhibition of tritiated thymidine incorporation, and 3) the effect of adrenalectomy 1 day before sacrifice on the spleen cell response to DM in vitro. The affinity of mouse spleen glucocorticoid receptors for [3H]DM is represented by a Kd at 4 C of ∼5 nM; in the guinea pig, the same tissue has a 20- fold lower affinity (Kd at 4 C, ∼0.1 μM). The concentrations of DM necessary for half-maximal inhibition of tritiated thymidine incorporation (mouse, 5 nM; guinea pig, 0.08 μM) closely approximate the half...

Studies of the mechanism of action of the tumour-inhibitory nitrosoureas

BCNU [1,3 bis-(2-chloroethyl)-1-nitrosourea] and some related nitrosoureas have been shown to have a wide spectrum of action against a number of transplanted rodent tumours. No correlation was found between the chemical instability of a nitrosourea and its antitumour activity. Unlike difunctional alkylating agents, the nitrosoureas inhibit the incorporation of tritiated precursors in DNA, RNA and protein to equal extents, the inhibition of tritiated thymidine incorporation into DNA occurring within 5 min of incubating cells with BCNU. Although the biological half life of BCNU was found to be very short (15 min by bioassay) a single injection was as effective against the established and widely-disseminated TLX5 lymphoma as against the early transplant. BCNU interfered specifically with the incorporation of labelled thymidine triphosphate into DNA, but no inhibition of DNA polymerase could be demonstrated at physiological dose levels. In their mechanism of action and in their biological properties the tumour inhibitory nitrosoureas are quite distinct from the bifunctional alkylating agents.

Studies on the physiological manifestations of cell mediated cytotoxicity: II. Inhibition of [ 3H]thymidine incorporation by plasmacytoma cells exposed in vitro to sensitized splenocytes

Splenocytes of both allogeneic and syngeneic origin, derived from mice challenged with a mineral oil induced BALB/c plasmacytoma (PCT), effected a marked and rapid inhibition of tritiated thymidine incorporation by the PCT cells in vitro . This inhibition was evident in the syngeneic confrontation even though exposure to the sensitized effector cells did not lead to detectable release of 51 Cr from labeled PCT target cells during 6 hr of incubation, a period of time sufficient to bring about appreciable chromium release when the spleen cells were allogeneic. The degree of inhibition of thymidine uptake caused by allogeneic effector cells was related to the effector/target cell ratio. Irradiation of the allogeneic splenocytes reduced or abolished their inhibitory capacity. Supernatants of interactions between PCT and sensitized allogeneic spleen cells had no specific inhibitory activity. It is suggested that inhibition of DNA precursor incorporation may offer a rapid and accurate means of assessing cytostatic effects mounted by sensitized effector cells.

Cyclic adenosine 3′:5′-monophosphate and inhibition of deoxyribonucleic acid synthesis in vitro

Results are presented which suggest that inhibition of tritiated thymidine incorporation into the deoxyribonucleic acid of bone marrow in vitro by high concentrations of cyclic adenosine 3':5'-monophosphate is pharmacological in nature, and thus apparently unrelated to the physiological actions of cAMP.