Altered sensitivity to colchicine and PHA in human cultured cells

Summary PHA-stimulated lymphocytes cultivated from a pair of human monozygotic twins yielded mostly tetraploid cells when colchicine was not used to arrest the metaphases. The rate of tetraploidy was also enhanced by colchicine in fibroblasts cultured without PHA. In in situ condition, larger than usual cells were observed. Other defects found in parental lymphocyte cultures included C-anaphase cells and increased cell aggregation. These results suggest a membrane mutation resulting in hypersensitivity to PHA and variant response to colchicine.     

Mitochondrial thymidine kinase and the enzymatic network regulating thymidine triphosphate pools in cultured human cells

In non-proliferating cells mitochondrial (mt) thymidine kinase (TK2) salvages thymidine derived from the extracellular milieu for the synthesis of mt dTTP. TK2 is a synthetic enzyme in a network of cytosolic and mt proteins with either synthetic or catabolic functions regulating the dTTP pool. In proliferating cultured cells the canonical cytosolic ribonucleotide reductase (R1-R2) is the prominent synthetic enzyme that by de novo synthesis provides most of dTTP for mt DNA replication. In non-proliferating cells p53R2 substitutes for R2. Catabolic enzymes safeguard the size of the dTTP pool: thymidine phosphorylase by degradation of thymidine and deoxyribonucleotidases by degradation of dTMP. Genetic deficiencies in three of the participants in the network, TK2, p53R2, or thymidine phosphorylase, result in severe mt DNA pathologies. Here we demonstrate the interdependence of the different enzymes of the network. We quantify changes in the size and turnover of the dTTP pool after inhibition of TK2 by RNA interference, of p53R2 with hydroxyurea, and of thymidine phosphorylase with 5-bromouracil. In proliferating cells the de novo pathway dominates, supporting large cytosolic and mt dTTP pools, whereas TK2 is dispensable, even in cells lacking the cytosolic thymidine kinase. In non-proliferating cells the small dTTP pools depend on the activities of both R1-p53R2 and TK2. The activity of TK2 is curbed by thymidine phosphorylase, which degrades thymidine in the cytoplasm, thus limiting the availability of thymidine for phosphorylation by TK2 in mitochondria. The dTTP pool shows an exquisite sensitivity to variations of thymidine concentrations at the nanomolar level.     

Deoxyribonucleoside triphosphate pools and differential thymidine sensitivities of cultured mouse lymphoma and myeloma cells

The effects of various concentrations of thymidine on DNA synthesis and deoxyribonucleoside triphosphate contents of a highly thymidine-sensitive cultured mouse lymphoma cell line (WEHI-7) and a relatively resistant mouse myeloma cell line (HPC-108) have been studied by 32P-labelling techniques. DNA synthesis in the myeloma cells was inhibited by thymidine at concentrations of 10(-3) M or greater, while DNA synthesis in the lymphoma cells was inhibited by concentrations 30-fold lower, consistent with the 25-fold difference between the two cell lines in sensitivity to growth inhibition by thymidine. Thymidine caused marked elevation of the dTTP and dGTP pools, slight elevation or no change in the dATP pool and a marked decrease in the dCTP pool in cells of both lines. The greater resistance of HPC-108 cells to thymidine inhibition was related to the finding that they normally contained a much higher concentration of dCTP than did the WEHI-7 cells. Pool size measurements on thymidine-treated (10(-4) M) cells of an additional seven sensitive lymphoma and six relatively resistant myeloma cell lines indicated that in all 15 lines studied, with one exception, a critical concentration of dCTP of about 32 nmol per ml of cell volume was required for the maintenance of normal rates of DNA synthesis. The dCTP content found normally in the lymphoma cells was only a little above this concentration. Amongst the myeloma lines, three contained similarly low levels of dCTP, but were more resistant to thymidine inhibition probably because of their inefficient production of dTTP from thymidine. Cells of the other four myeloma lines (including HPC-108) normally contained much higher dCTP concentrations. The mechanism of thymidine action was explained by reference to the known allosteric properties of ribonucleotide reductase.     

Interferon-specific cellular receptors in cultured human cells, differing in sensitivity to alpha-interferon

The interferon-specific cellular receptors in human cells cultures differing in sensitivity to alpha-interferon have been studied. The J-41 cells resistant to alpha-interferon are practically devoid of receptors highly specific to alpha-interferon. The coefficient of equilibrium and the number of receptors analyzed after Scatchard for J-96 culture of cells are 15.6 x 10(11) M-1 and 210 +/- 90, respectively. Evidently, resistance of J-41 cells to alpha-interferon is connected with the absence of interferon receptors and, as a consequence, inability to interferon-receptor interaction.     

Some factors affecting the sensitivity of cultured human cells to high-LET radiation

Summary Comparative effects of decay of DNA-bound¹²⁵I, of-radiation and of tritiated water on survival of the proliferative ability of cultured cells were examined. The results confirm a previous report that cells frozen to -196° C in the presence of 2M glycerol have lost a considerable proportion of their intracellular water. The data also suggest that the fraction of the lethal damage caused by deposition of radiation energy in intracellular water close to the DNA is greater for-radiation than for the decay of DNA-bound¹²⁵I.Inherited differences in the sensitivity of untransformed fibroblasts from individual humans to ionizing radiations and other DNA-damaging agents are being explored. The ratios of the sensitivities of various cell lines to particular agents can vary several-fold. Thus the RBE of various radiations is affected not only by the irradiation conditions and the water content of the cells but also by inherited abnormalities in the DNA repair systems in human cells.Dedicated to Prof. L.E. Feinendegen on the occasion of his 60th birthday     

Combination of thymidine and dexamethasone increases the K562 tumor cells sensitivity to human leukocytes cytotoxicity

The influence of the number of differentiating agents on sensitivity of human erythroleukemic cells K562 to human leukocyte-mediated non-MHC-restricted lysis was studied. It has been shown that a 4-day treatment of cells K562 by dexamethasone (1 microM) or phorbol-12-myristate-13-acetate (100 nM) leads to a significant decrease in sensitivity of the treated cells to non-specific lysis mediated by human leukocytes. On the contrary, the treatment of cells K562 by a combination of dexamethasone and thymidine (2 mM) leads to an increased sensitivity of the treated cancer cells to non-specific lysis mediated by the above effector cells, compared with the situation when these cells were treated by dexamethasone only. The treatment of cells K562 by a combination of thymidine and phorbol-12-myristate-13-acetate demonstrates a tendency (P < 0.1) to increase the sensitivity to non-specific lysis mediated by human leukocytes, as compared with the cases, when these cells were treated by phorbol ester only. It has been shown that the changes in K562 cell sensitivity to lytic action of leukocytes, under the chosen incubation time and doses of the used agents, well compare with the changes of erythroid differentiation of the cancer cells in the same conditions.     

Defective transport of thymidine by cultured cells resistant to 5-bromodeoxyuridine

A line of HeLa cells resistant to 5-bromo-2′-deoxyuridine (BUdR) was established by continuous culture in growth medium containing BUdR; during the selection period, BUdR concentrations, initially 15 μM, were gradually increased to 100 μM. Cells of a clone (HeLa/B5) established from this line were also resistant to 5-fluoro-2′-deoxyuridine (FUdR), but not to the free base, 5-fluorouracil. Although extracts of HeLa/B5 cells exhibited levels of thymidine kinase activity comparable to those of parental cells, rates of uptake of BUdR, FUdR, and thymidine into intact cells were much reduced. The kinetics of uptake of uridine and adenosine, nucleosides which appear to be transported independently of thymidine in HeLa cells, were similar for HeLa/B5 and the parental line (HeLa/0). Relative to thymidine uptake by HeLa/0 cells, that by HeLa/B5 cells was distinctly less sensitive to nitrobenzlthionosine (NBMPR), a specific inhibitor of nucleoside transport in various types of animal cells. Despite this difference in NBMPR sensitivity, both cell lines possessed the same number of high affinity NBMPR binding sites per mg cell protein. The altered kinetics of thymidine uptake and the NBMPR insensitivity of that function in HeLa/B5 cells suggest that resistance to BUdR is due to an altered thymidine transport mechanism.     

UV sensitivity in thymidine kinase deficient mouse erythroleukaemia cells

Wild type Friend erythroleukaemia cells (clone 707) and thymidine kinase (EC. 2.7.1.2.1) deficient derivatives were examined for sensitivity to killing by ultra-violet (UV) irradiation. Deficiency of thymidine kinase leads to increased cell killing as evidenced in all of twelve thymidine kinase deficient clones examined. A revertant thymidine kinase positive clone was found to have a level of thymidine kinase activity and UV sensitivity intermediate between wild type cells and its thymidine kinase deficient parent clone. The increased cell killing in thymidine kinase deficient clones is also reflected in increased mutagenesis by UV to 6-thioguanine resistance. It is suggested that the increased mutagenesis and sensitivity is due to defective DNA repair as a result of an imbalance in deoxyribonucleoside triphosphate pools in thymidine kinase deficient cells.     

Cytosolic thymidine kinase activity in cultured human fibroblasts from individuals with galactokinase deficiency

The structural genes for human galactokinase (GALK) and the human cytosolic form of thymidine kinase (TK1) are located on 17q21–q22. These two loci are tightly linked, and studies on Chinese hamster cell lines have shown that the expression of TK1 and GALK genes may alter simultaneously. We investigated the possibility of a dependent mutation of TK1 and GALK genes in cultured fibroblasts obtained from two patients homozygous for the GALK^G-deficient gene. Since we showed that the TK1 level varies as a function of the passage and the growth rate of a given strain, our experiments were performed on nonstored skin fibroblasts, between the third and the fifth passage for both controls and patients. We found that TK1 levels in GALK-deficient cells were almost 75% of those observed in control strains with a similar growth rate. Previous results in the literature have shown a pronounced decrease in TK1 activity in three GALK-deficient fibroblastic strains. We suggest that these disparities of TK1 levels in GALK-deficient fibroblasts may be related either to genetic heterogeneity of GALK deficiency or to differences in culture conditions. This work was supported in part by grants from La CNAMTS and l’Université de Paris-Sud (AI 86 10).     

Interferon-gamma reduces the sensitivity of cultured and fresh human tumor cells to lysis by lymphokine-activated killer cells

We have shown that interferon-gamma (IFN-gamma), in pharmacologically achievable doses, can reduce the the sensitivity of human tumor cells to lysis by allogeneic lymphokine-activated killer (LAK) effector cells. Cultured tumor cells showed a consistent reduction in sensitivity to lysis following pretreatment for 18 h with 1-10 units/ml IFN-gamma. Tumor cells cultured up to 7 days in 100 units/ml IFN-gamma remained less sensitive to lysis. Induction of protection from LAK did not appear to correlate with IFN-gamma-induced changes in cell growth or proliferation. Reduced LAK sensitivity also did not correlate with the level of expression of major histocompatibility antigens. Eight of 11 surgically obtained human tumor cell specimens showed a reduction in sensitivity to lysis by allogeneic LAK cells following pretreatment with IFN-gamma. IFN-induced reduction of tumor cell sensitivity to lysis by LAK may play a role in altering the host-tumor relationship, since relatively high concentrations of IFN-gamma may exist in the tumor microenvironment.     

The pyrimidine nucleotide carrier PNC1 and mitochondrial trafficking of thymidine phosphates in cultured human cells

In cycling cells cytosolic de novo synthesis of deoxynucleotides is the main source of precursors for mitochondrial (mt) DNA synthesis. The transfer of deoxynucleotides across the inner mt membrane requires protein carriers. PNC1, a SLC25 family member, exchanges pyrimidine nucleoside triphosphates in liposomes and its downregulation decreases mtUTP concentration in cultured cells. By an isotope-flow protocol we confirmed transport of uridine nucleotides by PNC1 in intact cultured cells and investigated PNC1 involvement in the mt trafficking of thymidine phosphates. Key features of our approach were the manipulation of PNC1 expression by RNA interference or inducible overexpression, the employment of cells proficient or deficient for cytosolic thymidine kinase (TK1) to distinguish the direction of flow of thymidine nucleotides across the mt membrane during short pulses with [(3)H]-thymidine, the determination of mtdTTP specific radioactivity to quantitate the rate of mtdTTP export to the cytoplasm. Downregulation of PNC1 in TK1(-) cells increased labeled dTTP in mitochondria due to a reduced rate of export. Overexpression of PNC1 in TK1(+) cells increased mtdTTP pool size and radioactivity, suggesting an involvement in the import of thymidine phosphates. Thus PNC1 is a component of the network regulating the mtdTTP pool in human cells.     

Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells.

Treatment of Ltk^?, mouse L cells deficient in thymidine kinase (tk), with Bam I restriction endonuclease cleaved DNA from herpes simplex virus-1 (HSV-1) produced tk⁺ clones with a frequency of 10^?6/2 μg of HSV-1 DNA. Untreated cells or cells treated with Eco RI restriction endonuclease fragments produced no tk+ clones under the same conditions. The thymidine kinase activities of four independently derived clones were characterized by biochemical and serological techniques. By these criteria, the tk activities were found to be identical to HSV-1 tk and different from host wildtype tk. The tk⁺ phenotype was stable over several hundred cell generations, although the rate of reversion to the tk^? phenotype, as judged by cloning efficiency in the presence of bromodeoxyuridine, was high (1–5 × 10^?3). HSV-1 DNA Bam restriction fragments were separated by gel electrophoresis, and virtually all activity, as assayed by transfection, was found to reside in a 3.4 kb fragment. Transformation efficiency with the isolated fragment is 20 fold higher per gene equivalent than with the unfractionated total Bam digest. These results prove the usefulness of transfection assays as a means for the bioassay and isolation of restriction fragments carrying specific genetic information. Cells expressing HSV-1 tk may also provide a useful model system for the detailed analysis of eucaryotic and viral gene regulation.     

Two-dimensional protein profiles of cultured stromal and epithelial cells from hyperplastic human prostate

Studies were undertaken to compare and contrast the two-dimensional protein profiles of epithelial and stromal cells from hyperplastic human prostate to establish the protein composition of the two major cellular components of the prostate. Epithelial and stromal cells were isolated from human prostate obtained from patients undergoing open prostatectomy for benign prostatic hyperplasia (BPH). Proteins, isolated from the two cell populations and separated by two-dimensional (2D) electrophoresis, were analyzed by silver staining, fluorography of [35S]-methionine-labeled proteins, and immunoprotein blotting. Isolated prostatic epithelial cells, but not stromal cells, contained cytokeratin polypeptides 5, 6, 7, 8, 13, 14, 15, 16, 17, 18, and 19. Although vimentin could not be identified in silver stained 2D gels and fluorographs of cultured prostatic epithelial cells, a low level of immunoreactivity was noted following immunoblot analysis of epithelial cells proteins by the use of an anti-vimentin polyclonal. Vimentin was prominently expressed in cultured prostatic stromal cells and could be identified on silver stained 2D gels, fluorographs, and immunoblots of stroma-derived proteins. In addition, stromal marker proteins SM1, SM2, and SM3 were identified in 2D gels of stromal cells to distinguish them from epithelial cells. These studies demonstrate (1) the two-dimensional protein profile and cytokeratin polypeptide composition of cultured epithelial cells from hyperplastic human prostate and (2) the 2D protein profile of cultured prostatic stromal cells and identification of specific stromal marker proteins.     

High sensitivity of cultured human trophoblasts to ribosome-inactivating proteins.

Many plant proteins possessing abortifacient activities were identified as ribosome-inactivating proteins (RIPs). The effect of several ribosome-inactivating proteins (saporin 6, dianthin 32, pokeweed antiviral protein from seeds, gelonin, bryodin-R, and momordin) on primary cultures of human trophoblasts and human embryonal fibroblasts and on choriocarcinoma (JAR and BeWo) and ovarian carcinoma (TG) cell lines was studied. Protein synthesis of human trophoblasts and BeWo cells was lowered by RIPs more than that of other cells. The trophoblastic receptors for estradiol were not affected by treatment of the cells with momordin. The binding and uptake of saporin 6 and momordin by BeWo and HeLa cells were not correlated to cell toxicity.     

Description and analysis of differential sensitivity to glucocorticoids in Fao cells

This study shows that the derived hepatoma cell line Fao displays different sensitivities for glucocorticoid induction of tyrosine aminotransferase (TAT), alanine aminotransferase (AAT) and gamma-glutamyltransferase (GGT). This was seen in the different behaviors of nine steroids with respect to these three effects: (1) in the presence of full agonists (dexamethasone or deacylcortivazol), half-maximal induction of GGT occurred at approx 5- to 6-fold higher agonist concentrations than those required for half-maximal induction of AAT and TAT; (2) in the presence of full antagonists (RU 486, R5020, or progesterone) the GGT response induced by an equal agonist concentration was inhibited at concentrations approx 4- to 5-fold lower than those required for an equivalent inhibition of TAT response; (3) in the presence of cortexolone, deoxycorticosterone, 11 beta-hydroxyprogesterone and dexamethasone-3'-oxetanone, there was a partial agonistic effect (30-50%) on TAT and AAT responses, whereas there was a mainly antagonistic effect (very weak agonistic effect: 0-10%) on GGT response; (4) regardless of the steroid or its full or partial agonist activity, a given TAT induction level (50%, for example) always corresponded to the same AAT and GGT induction levels (50 and 10% respectively). We provide evidence showing that the three above-mentioned biological responses are mediated via the same type of glucocorticoid receptor binding site. Consequently, this differential behavior probably originates from a phenomenon occurring after the common steps (activation, translocation) that follow the formation of the steroid-receptor complex. This leads us to propose a model in which this phenomenon is assumed to originate from a difference in the affinities of the activated receptor for the nuclear acceptor sites of the TAT and GGT genes.