Inhibition Of Cell Multiplication Research Articles

Further evidence for a cell surface proteinase essential to the growth of cultured fibroblasts

Specific antibodies and protein proteinase inhibitors will inhibit cell-surface proteinase activity on human fibroblasts and cause a concomitant inhibition of DNA synthesis and of cell multiplication. An insolubilized proteinase inhibitor also inhibits cell multiplication. The same reagents partially inhibit the multiplication of mouse L cells, both in monolayer and suspension culture, and inhibit the mitogenic effect of epidermal growth factor (EGF) on both types of cell.

Plasma membrane as target of alkylating agents

N mustard resistant Walker cells exhibit the same frequency of DNA interstrand cross-links and the same rate of cross-link removal as the sensitive parental line. Employing cytostatically active concentrations of chlorambucil covalently bound to polyethyleneimine, the extent of DNA cross-linking is reduced to levels observed in the presence of nontoxic concentrations of free chlorambucil. It is concluded, therefore, that DNA cross-links alone are not sufficient to explain the inhibition of cell multiplication by alkylating agents and that additional mechanisms have to be considered. Evidence for an interference of alkylating agents with several enzymes of the plasma membrane is presented. An inhibition by N mustard of the furosemide-sensitive Na +/K +/Cl −-cotransport and the Na +/H +-antiport is described in greater detail. Considering the fact that the enzymes which are affected by alkylating agents are controlled by growth factors it was investigated whether a synergism between inhibitors of early growth-factor-controlled reactions and alkylating agents is to be seen. It is demonstrated that mepacrine, an inhibitor of phospholipase C, and the calmodulin binding drugs, chloropromazine and flunarizine, amplify the action of N mustard.

Metabolism of cytosine arabinoside in Tetrahymena pyriformis.

The metabolism of cytosine arabinoside (araC) in Tetrahymena pyriformis amicronucleate strain W was studied. araC inhibited cell multiplication and protein synthesis at concentrations higher than 0.1 and 0.25 M respectively. araC had no effect on protein synthesis. araC was converted to araCMP, araCDP and araCTP by homogenized cell preparations. A deaminase activity converted araC to uracil arabinoside. The deaminase activity totally inhibited by tetrahydrouridine (THU) at a concn of 4 X 10(-6) M. The Ki for THU was 8 X 10(-8) M.

Effect of cinnamoyl putrescines on in vitro cell multiplication and differentiation of tobacco explants

Hydroxycinnamoyl putrescines promote the cell multiplication of leaf discs of a tobacco mutant, RMB7, cultivatedin vitro on the Murashige and Skoog medium. This mutant never accumulates these molecules during its development and does not enter in floweirng. Maximal effect is obtained at 2.5·10(-4)M. The same molecules inhibit bud formation ofNicotiana tabacum var. Xanthi nc, at 5·10(-5) M but promote callus formation. From 10(-4) M to 5·10(-3) M they strongly inhibit cell multiplication and bud formation without toxic effect. Their possible role in plant metabolism is discussed.

Effects of ascorbic and dehydroascorbic acid on the multiplication of tumor ascites cells in vitro.

The effects of AA and DHA on ATP C+ cell multiplication in vitro were studied by measuring incorporation of 3H thymidine into DNA. The results obtained demonstrate that both AA and DHA have the same effects: they favor cell multiplication at low doses and inhibit it at high doses. Experiments carried out with serial doses of both these substances revealed that AA is more efficient in determining both stimulating and inhibiting effects. The lesser efficiency of DHA may be attributed to its limited stability in culture medium. Studies on the effect of high doses of AA and DHA added to the culture medium in single or fractionated doses revealed that fractionated administration is more efficient in inhibiting cell multiplication than single administration.

Synergistic antiviral and antiproliferative activities of Escherichia coli-derived human alpha, beta, and gamma interferons.

The antiviral and antiproliferative effects of highly purified Escherichia coli-derived human interferons (IFNs) were examined in human melanoma cells (Hs294T). Antiproliferative activity was monitored by measuring inhibition of cell multiplication, and antiviral activity was determined by inhibition of herpes simplex virus type 1 replication. Treatment of cells with IFN-gamma in combination with IFN-alpha A or IFN-beta 1 resulted in potentiation of both antiproliferative and antiviral activities. In contrast, combination treatments composed of IFN-alpha A and IFN beta 1 yielded inconsistent results. Some combinations reflected additive responses, whereas others were antagonistic. To examine correlations between IFN-induced biological activities and interactions of the different IFNs with cell surface receptors, in vivo [35S]methionine-labeled IFN-alpha A was prepared. Binding studies indicated the presence of 2,980 +/- 170 receptors per cell, each with an apparent Kd of (8.4 +/- 1.3) X 10(-11) M. Results from competitive binding studies suggested that Hs294T cells possess at least two types of IFN receptors: one which binds IFN-alpha A and IFN-beta 1 and another to which IFN-gamma binds.

Dual effects of estradiol on normal and tumor pituitary cell multiplication

We have compared the effects of estradiol on the [ 3H]thymidine (TdR) incorporation into the DNA of 2 rat tissues whose growth is controlled by estradiol in vivo in 2 opposite directions: the normal anterior pituitary and the MtF 4 pituitary tumor transplanted under the kidney capsule. Small pieces of pituitary or tumor from Fischer rats, treated or not by estradiol in silastic tubing, were incubated in vitro with [ 3H ]TdR. The [ 3H]TdR incorporated per μg DNA was decreased in tumor after 2 to 8 day-estradiol treatment while simultaneously, in the same rats, it was increased in the pituitary. In addition, we studied the effect of estradiol in vitro on the F 4C 1 cell line obtained from the MtF 4 tumor. A dose-dependent decrease of both the [ 3H ]TdR incorporated into DNA and the DNA amount was observed between 10 −6 and 10 −5 M estradiol. These results suggest that the control of the pituitary or MtF 4 tumor growth by estradiol in vivo is in part due to an inhibition of cell multiplication. Although estradiol inhibits the growth of a clone of MtF 4 tumor cells in vitro we cannot decide whether or not the in vivo effect of estradiol is direct.

Efficiency of various bile salt preparations for stimulation of Clostridium difficile spore germination.

Taurocholate, desoxycholate, and cholate stimulated germination of Clostridium difficile spores in broth medium and enhanced recovery of C. difficile spores on a selective agar medium. Desoxycholate and some crude taurocholate preparations also inhibited multiplication of vegetative cells. At a concentration of 1.2 X 10(-2) M, sodium cholate inhibited multiplication of vegetative cells, but at concentrations of 1.2 X 10(-3) to 2.4 X 10(-3) M, it stimulated germination without inhibiting cell multiplication. Thus, pure sodium taurocholate and sodium cholate may effectively be incorporated in cefoxitin-cycloserine-fructose agar, whereas some crude preparations of sodium taurocholate decrease recovery on this medium.

Binucleation and abnormal chromosome distribution inEuglena gracilis cells treated with dimethyl sulfoxide

The addition of 5% of dimethyl sulfoxide (DMSO) to cultures ofEuglena gracilis in the logarithmic phase of growth caused an immediate inhibition of cell multiplication and motility without completely blocking nuclear division. Importantly, some 50% of the cells were 2–3 times larger than normal and were also binucleate after 24–48 hours of treatment. Evidently binucleation resulted from the lack of cytokinesis after mitosis was induced. Transmission electron microscopy, using serial sections, showed the occurrence of nuclei either with a normal or a reduced number of chromatin masses. Solvent withdrawal led to a rapid recovery of all the normal cell activities.

Indomethacin and Aspirin Do Not Inhibit the Antiviral or Anti-proliferative Actions of Interferon

Neither indomethacin nor aspirin, at concentrations which inhibited the formation of prostaglandins and prevented the interferon-induced increase in the intracellular concentration of cyclic GMP, had any significant effect on the development of the interferon-induced antiviral state either in mouse L1210 cells challenged with vesicular stomatitis virus or in mice infected with encephalomyocarditis virus. Furthermore, neither drug had any significant effect on the interferon-induced inhibition of cell multiplication in cultures of mouse leukaemia L1210 cells. The differences in the effects of these cyclo-oxygenase inhibitors on different interferon effects may provide some insight into the different pathways of interferon action.

Conditions Supporting Repair of Potentially Lethal Damage Cause a Significant Reduction of Ultraviolet Light-Induced Division Delay in Synchronized and Plateau-Phase Ehrlich Ascites Tumor Cells

Repair of potentially lethal damage (PLD) induced by uv light in synchronized and in plateau-phase cultures of Ehrlich ascites tumor cells was studied by measuring cell survival. In particlar the influence of conditions supporting repair of PLD on growth kinetics was investigated. In synchronized G/sub 1/, S, or G/sub 2/ + M cells as well as in plateau-phase cells, uv light induced, almost exclusively, delay in the next S phase. A significant decrease of this delay was observed when the cells were incubated for 24 hr in balanced salt solution. Repair of PLD after uv irradiation was found to occur in plateau-phase cells and in cells in different phases of the cell cycle provided that after irradiation these were kept under conditions inhibiting cell multiplication (incubation in balanced salt solution or in conditioned medium). The repair time constant t/sub 50/ was significantly higher than those found for X irradiation (5-10 hr compared to 2 hr), and repair was not significantly inhibited by either 20 ..mu..g/ml cycloheximide or 2 mM caffeine in 24 hr.

Adaptation of the human breast cancer cell line MCF-7 to serum free medium culture on extracellular matrix

The human breast cancer cell line MCF-7 has been adapted to grow in serum free medium on an extracellular matrix produced by bovine corneal endothelial cells. The cells have been continuously maintained in these conditions for more than 11 months with the same doubling time as the original population grown on plastic dishes in presence of charcoal treated fetal calf serum. The hormonal control of their growth appears intact. Insulin and epidermal growth factor stimulate cell proliferation. Estradiol-17β (E 2 ), dexamethasone, L-triiodothyronine, prolactin have no effect. Treatment with tamoxifen and estrone induces an inhibition of cell multiplication. This inhibition by tamoxifen can be rescued by 4 % charcoal treated fetal calf serum and E 2 but not by E 2 alone.

Rapid increase in guanosine 3′,5′-cyclic-monophosphate in interferon-treated mouse leukemia L1210 cells: Relationship to the development of the antiviral state and inhibition of cell multiplication

Treatment of mouse leukemia L1210 cells with electrophoretically pure mouse interferon caused a rapid (1 to 5 min) and transitory (duration 5 to 10 min) increase in the intracellular concentration of guanosine 3′,5′-cyclic monophosphate (cyclic GMP). No significant increase in the intracellular concentration of adenosine 3′,5′-cyclic monophosphate (cyclic AMP) was observed until 2 to 3 hr after the addition of interferon. Electrophoretically pure mouse interferon caused a dose-dependent increase in the intracellular concentration of cyclic GMP at concentrations ranging from 1.0 to 10 4 unite/ml. The interferon-induced increase in cyclic GMP was abrogated by specific antibody to mouse interferon. Furthermore, interferon had no effect on the intracellular concentration of cyclic GMP in a line of L1210 cells resistant to the action of interferon. The interferon-induced increase in cyclic GMP was calcium dependent and was inhibited by indomethacin and aspirin at concentrations of 10 −7 to 10 −6 M. Depletion of intracellular calcium prior to the addition of interferon abrogated the interferon-induced increase in cyclic GMP without preventing either the development of the antiviral state or the inhibition of cell multiplication in interferon-treated L1210 cells. These results suggest that cyclic GMP may not mediate these two biologic effects of interferon but may rather reflect rapid changes in interferon-treated cells.

Calcitonin stimulates plasminogen activator in porcine renal tubular cells: LLC-PK1.

Plasminogen activators are highly selective proteases that activate the proenzyme plasminogen to the general protease, plasmin. We studied a porcine kidney cell line, originally isolated as a high producer of plasminogen activator, in which activities of cellular adenylate cyclase and cAMP-dependent protein kinase are increased in response to calcitonin. We found that salmon calcitonin, in the concentration range 0.03-300 nM, increased plasminogen activator production up to approximately 1,000-fold and concurrently inhibited cell multiplication; both of these effects were reversible. Human calcitonin was approximately 0.01 times as potent as salmon calcitonin, corresponding to potency differences observed in other biological systems. Plasminogen activator production was also stimulated by other agents that raise cellular cAMP levels such as cholera toxin, phosphodiesterase inhibitors, and vasopressin, but not to the same extent as by calcitonins. The rapidity and sensitivity of the plasminogen activator determination and other cellular responses may make it possible in the future to use this cell stain in a convenient bioassay for calcitonins and their analogues.

Effects of the mycotoxin botryodiplodin on mammalian cells in culture

The effects of botryodiplodin, a mycotoxin synthesized by Botryodiplodia theobromae and by some strains of Penicillium roqueforti. The toxin inhibited cell multiplication in growing cultures at concentrations which were without toxic effect on cultures nearing or at confluence. In growing cultures the toxin affected DNA, RNA and protein synthesis, the effect on DNA synthesis being the greatest. The removal of mycotoxin from the culture medium brought about partial recovery of the synthetic activities of the cells. The implications of the results with respect of the recovery of DNA synthesis are discussed.

Reversibility of the transformed and neoplastic phenotype. I. Progressive reversion of the phenotype of X-ray-transformed C3H/10T1/2 cells under prolonged treatment with interferon.

X-ray transformed mouse C3H/10T1/2 cells were cultivated and passaged in the continuous presence of partially purified mouse interferon. This prolonged interferon treatment resulted in a stepwise progressive reversion of the transformed phenotype to the non-transformed phenotype. Thus interferon-treated cells displayed an epithelioid morphology, grew to a lower cell density, and were no longer tumorigenic. Reversion to the non-transformed phenotype was, however, stable only as long as interferon was continuously present in the culture medium. When interferon was removed, the cells "back reverted" to the transformed phenotype. Our results suggest that interferon induced a reversion of the transformed phenotype in the entire cell population rather than a selection of an interferon resistant cell population. C-type viral particles and significant levels of reverse transcriptase were present in transformed cells, but neither present in the parental 10T1/2 cells nor in interferon-treated cells. When interferon was removed form the culture medium, viral particles and reverse transcriptase activity were again detected. It is possible, therefore, that interferon induces reversion through its antiviral activity, or that it induces reversion by its effects on cell function and structure, independently of any antiviral effect. Inhibition of cell multiplication per se does no appear to be sufficient to induce reversion, since cycloheximide inhibited cell multiplication; however, even after ten passages, it did not affect tumorigenicity. Our results suggest the possibility that interferon may act in vivo not only by inhibiting tumor cell multiplication but also by inducing a reversion. Patients with some tumors may therefore benefit from long-term interferon treatment.

Antiproliferative and antiviral activities of human leukocyte interferons

The antiproliferative effects of purified human leukocyte interferons were measured by their ability to inhibit cell multiplication of the human lymphoblastoid Daudi cell line. The growth-inhibitory and antiviral activities fractionate together throughout the purification of leukocyte interferon. In all cases, those fractions which exhibited antiviral activity also inhibited proliferation of Daudi cells. The final stage of protein purification, high-performance liquid chromatography on octyl-silica particles (pH 4.0), separated at least eight species of leukocyte interferon, all of which possessed growth-inhibitory activity. Because these two biological activities purify together and because the homogeneous species exhibit both activities, we conclude that the antiproliferative and the antiviral activities are both intrinsic properties of these human leukocyte interferons. However, the ratio of the growth-inhibitory to antiviral activity was not constant among the species. In addition, the ratio of the antiviral activity on human cells to that on bovine cells varied from species to species. We conclude that the individual members of the family of human leukocyte interferon proteins differ in structure and that these structural differences manifest themselves as variations in their intrinsic biological activities.

Induction of sister chromatid exchanges in human diploid fibroblasts by mutagens with and without rat liver microsomal activation.

The frequency of sister chromatid exchange (SCE) in WI-38 cells was estimated by the 5-bromodeoxyuridine (BrdUrd)-dye technique after one hour's exposure to cyclophosphamide (CY), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitroquinoline-1-oxide (4NQO), and maleic hydrazide (MH) with and without the addition of rat liver microsomal suspension (S-9) fraction with cofactors (S-9 mix). CY at concentrations from 1 x 10(-5) M to 1 x 10(-3) M with S-9 mix increased the number of SCEs per cell in a dose-dependent manner. Without S-9 mix, CY at concentrations below 1 x 10(-3) M failed to produce more SCEs than the controls. MNNG at 1 x 10(-8) M and 4NQO at 1 x 10(-7) M without S-9 produced significant increases in SCEs per cell. Addition of S-9 during treatment slightly decreased the effects of MNNG and 4NQO in the formation of SCEs. MH was tested at pH 6.4 and pH 7.6. At pH 7.6, MH at 1 x 10(-3)M without S-9 mix inhibited cell multiplication, but did not cause a significant increase of SCEs per cell. There were no interactions between MH (2 x 10(-4) M) and S-9 mix nor between MH and the pH levels tested. These results indicate that in the presence of metabolic activation, SCE formation in human diploid fibroblasts in vitro may be used as a potential assay for mutagenicity.

Selenium-containing tRNAs from Clostridium sticklandii: cochromatography of one species with L-prolyl-tRNA.

75Se-Labeled tRNAs were synthesized by Clostridium sticklandii cultures supplemented with 1 microM sodium [75Se]selenite or [75Se]selenocysteine. This process is highly specific for selenium; it occurred in the presence of 1.2 mM sodium sulfide and was not decreased by the further addition of a 500-fold molar excess of cysteine. The 75Se in these tRNAs was located in the polynucleotide portion of the molecules and not in esterified (alkali-labile) selenocysteine. Inhibition of cell multiplication by antibiotics that block either protein synthesis or DNA-dependent RNA synthesis did not prevent this 75Se incorporation. Three [75Se]tRNAs were separated from C. sticklandii cells labeled in the presence of chloramphenicol and were partially purified by chromatography on benzoylated DEAE-cellulose and DEAE-Sephadex A-50 columns. These were designated seleno-tRNAs I, II, and III according to their elution sequence from benzoylated DEAE-cellulose. Cochromatography of purified seleno-tRNA II on DEAE-Sephadex A-50 with an L-proline-accepting species suggests that it is a selenium-containing L-prolyl-tRNA.

Extrusion of DNA from the macronucleus of Tetrahymena pyriformis GL.

Administration of the thymidine analog 5-bromodeoxyuridine to exponentially growing cultures of Tetrahymena pyriformis GL in chemically defined medium results in inhibition of cell multiplication by at least one generation before DNA synthesis stops. Cell multiplication can be restored in these cultures, if they are transferred to fresh growth medium, but although most of the cells in the culture contain close to a G2-amount of DNA, a full DNA replication round is a prerequisite for renewed cell multiplication. Large extrusion bodies are found at the first division after transfer to fresh growth medium. Autoradiographic analysis has revealed that the DNA in the extrusion body is a representative of the DNA in the macronucleus indicating a random distribution of DNA between daughter nuclei and extrusion body.