Effect of treatment with azathioprine on the responses of rat lymphocytes to phytohaemagglutinin.

The proliferative responses of rat peripheral blood lymphocytes (PBL) and spleen cells to phytohaemagglutinin (PHA) were studied after single or multiple (daily for 4 days) injections of azathioprine (AZ). Lymphopenia developed within 4 h of a single dose (78 mg/kg) of AZ and persisted for at least 72 h. There was no lymphopenia 24 h after the last of 4 daily injections. In vitro, PBL were more sensitive than spleen cells to the inhibitory effect of AZ. Likewise, the responses of PBL were relatively more depressed than those of spleen cells after single or multiple injections of AZ. The degree of depression was less than was expected from the effect of AZ in vitro. Multiple small doses were more depressive than multiple large doses. Serum from treated rats, used at 20% concentration, was more depressive than normal. Thus, rat lymphocytes are quite sensitive to AZ in vitro, but appear to be relatively resistant in vivo, this resistance resembling the resistance of the primary antibody response to AZ treatment.     

Desensitization of the vascular contractile response to cumulative doses of alpha 2-adrenoceptor agonists

Contractile responses to single or cumulative doses of alpha-adrenoceptor agonists were compared in the tail artery and the saphenous vein of the rat. In the rat tail artery, there were no differences in the dose-response relationships to noradrenaline, methoxamine, and KCl whether the agonists were applied as single or cumulative doses. However, the responses to single doses of clonidine and B-HT 920 were significantly larger than similar doses applied cumulatively. In the rat saphenous vein, responses to single doses of noradrenaline, clonidine, and B-HT 920 were also significantly larger than the corresponding cumulative doses. However, there was no difference in the responses to KCl. It was suggested that desensitization of alpha 2-adrenoceptors in these vessels may result in the diminished responses to cumulative doses of the agonists. Desensitization appeared to be specific to alpha 2-adrenoceptors, since the effect was not observed in responses mediated by the alpha 1-adrenoceptors and KCl.     

Effects of single and split doses of cobalt-60 gamma rays and 14 MeV neutrons on mouse stem cell spermatogonia

The long-term effects of ionizing radiation on male gonads may be the result of damage to spermatogonial stem cells. Doses of 10 cGy to 15 Gy (60)Co gamma rays or 10 cGy to 7 Gy 14 MeV neutrons were given to NMRI mice as single or split doses separated by a 24-h interval. The ratios of haploid spermatids/2c cells and the coefficients of variation of DNA histogram peaks as measures of both the cytocidal and the clastogenic actions of radiation were analyzed by DNA flow cytometry after DAPI staining. The coefficient of variation is not only a statistical examination of the data but is also used here as a measure of residual damage to DNA (i.e. a biological dosimeter). Testicular histology was examined in parallel. At 70 days after irradiation, the relative biological effectiveness for neutrons at 50% survival of spermatogonial stem cells was 3.6 for single doses and 2.8 for split doses. The average coefficient of variation of unirradiated controls of elongated spermatids was doubled when stem cells were irradiated with single doses of approximately 14 Gy (60)Co gamma rays or 3 Gy neutrons and observed 70 days later. Split doses of (60)Co gamma rays were more effective than single doses, doubling DNA dispersion at 7 Gy. No fractionation effect was found with neutrons with coefficients of variation.     

Experimental models of immunochemotherapeutic synergism: study of the influence of the treatment schedule

Graded doses of LSTPA or L1210 leukemia cells were injected ip or iv into fully compatible hosts or mice incompatible for Multiple Minor Histocompatibility Loci (MMHL). Three days later the animals were treated with single doses of BCNU, NM, DTIC and VCR. The results showed that NM and VCR could synergize with the weak anti-tumor immune responses of MMHL-histocompatible mice only upon ip injection of the tumor. If the same tumor has been injected iv, only BCNU could synergize with the host's antitumor response. On DTIC treatment, no synergistic effects were detectable for either route of tumor's challenge.     

Changes in hepatocytes and the functional activity of the adrenal cortex in rats following administration of the pigment anthraquinone violet

The response of hepatocytes and cells of adrenal cortex of males of white rats to a single introduction per os of sunflower oil or anthraquinone violet pigment (AVP), routinely used for the painting of plastics, was evaluated. 5 ml of sunflower oil or of AVP, prepared as oil suspension, were fed. After 1 or 24 hour introductions of oil, an increase in dividing ability of hepatocytes was observed, whereas after 6 or 72 hour introductions no difference from the control was registered. The introduction of oil caused a decrease of the functional activity of cells in fasciculata zone of adrenal cortex after 1, 6, 24 and 72 hours, as judged from changes in the nuclear volume. AVP in doses of 5.0 and 1.2 g/kg intensified hepatic polyploidy after 1 and 24 hours as compared with rats administered only the oil. After 6 and 72 hours, no difference from the control was registered. In the fasciculata zone of adrenal cortex, AVP in both the doses employed increased the functional activity of cells after 1, 6 and 24 hours. The nature of the response of hepatic cells on the introduction of the above substances presumably depends on the degree of adrenal cortex functional activity.     

Bleomycin, in contrast to gamma irradiation, induces extreme variation of DNA strand breakage from cell to cell

Chinese hamster ovary cells grown in vitro were treated with bleomycin or irradiated with high doses of 60Co gamma rays (200 and 400 Gy). DNA strand breaks in single cells were analysed by using our newly introduced microelectrophoretic technique. Bleomycin seems to act in a selective manner so that in some cells the DNA is heavily degraded while in others there is only moderate or no measurable damage. In contrast, a uniform response was found after gamma irradiation. To achieve the same magnitude of DNA fragmentation as in the most severely bleomycin-damaged cells, irradiation with more than 200 Gy is required. Some 8000 double-strand breaks per cell are produced by 200 Gy which will convert the molecular weight of the DNA to the range of 10(8)-10(9) dalton, and free migration of DNA fragments occurs during electrophoresis. We include also a detailed study of the DNA migration pattern following doses of 0-100 Gy gamma rays.     

Development of resistance in Fisher lymphadenosis cells to dipin and bruneomycin when these preparations are used separately and in combination]

It was shown that dipin and bruneomycin resistant tumor cells appeared in mice with transplanted lymphadenosis after 10 passages on single use of the drugs. When the drugs were used in combination, no lymphadenosis cells resistant either to bruneomycin, or to dipin and their combination were found. The combined use of the drugs prevented development of resistance to them in the lymphadenosis cells at least during 10 passages on mice. The data on the possible prevention of the resistance development in the mouse lymphadenosis cells by means of combined use of low doses of dipin and bruneomycin provided an assumption that it is expedient to test the combination in clinics.     

Tumor immunity directed against MOPC 104E: Effect of various therapeutic regimens

Summary Animals bearing the passable plasmacytoma MOPC 104E could be cured of palpable tumors (0.6–2.0×10⁸ cells) with single 10–250 mg/kg doses of cyclophosphamide or single localized x-ray doses greater than 1600 R. Residual tumor immunity of cured animals was determined by their ability to reject graded numbers of viable MOPC 104E cells 30 days following curative therapy. High doses of cyclophosphamide (250 mg/kg), although curative, left significantly less residual tumor immunity than either low dose cyclophosphamide (10 mg/kg) or localized irradiation. Animals cured of palpable tumors by high doses of cyclophosphamide nonetheless rejected greater numbers of cells in secondary challenge than did untreated control animals.This investigation received support from NIH Grants 13371, 17065, 05136, and 09082 from the National Cancer InstituteSubmitted in partial fulfilment of the degree Doctor of Philosophy in Radiation Biology     

Absence of a dose-fractionation effect on neoplastic transformation induced by fission-spectrum neutrons in C3H 10T1/2 cells

We have investigated the effect of fission-spectrum neutron dose fractionation on neoplastic transformation of exponentially growing C3H 10T1/2 cells. Total doses of 10.8, 27, 54, and 108 cGy were given in single doses or in five equal fractions delivered at 24-h intervals in the biological channel of the RSV-TAPIRO reactor at CRE-Casaccia. Both cell inactivation and neoplastic transformation were more effectively induced by fission neutrons than by 250-kVp X rays. No significant effect on cell survival or neoplastic transformation was observed with split doses compared to single doses of fission-spectrum neutrons. Neutron RBE values relative to X rays determined from data for survival and neoplastic transformation were comparable.     

Cytogenetic and dominant lethal studies on captan.

Possible mutagenic activity of captan was investigated by in vitro and in vivo cytogenetic studies and by the dominant lethal study in mice. In vitro cytogenetic study with cultured human diploid cells revealed a significant increase in the frequency of cells showing stickiness and a severe mitotic inhibition at concentrations of 3.0 and 4.0 microgram of captan per ml. although no chromosomal aberrations were observed. In in vivo cytogenetic study, no chromosomal aberrations were induced in the bone marrow cells of rats treated orally with captan at a single dose of 500, 1000 or 2000 mg/kg or at five consecutive doses of 200, 400 or 800 mg/kg/day. Dominant lethal study also failed to show any mutation induction after treatment of male mice with daily oral dose of 200 or 600 mg of captan per kg bw for five days.     

Genotoxicity, metabolism and blood kinetics of epichlorohydrin in mice

Epichlorohydrin (ECH), a direct mutagen in vitro, did not induce chromosomal aberrations in bone-marrow cells of CD1 mice given single oral doses of 50 and 200 mg/kg in water. The ECH diol derivative (3-chloro-1,2-propanediol) was tested in vitro by a forward-mutation assay on the yeast Schizosaccharomyces pombe and showed a weak but significant mutagenic effect. The failure of ECH to induce mutagenic effects appears to be due to the rapid metabolic clearance of the compound in vivo. ECH blood kinetics at both doses, and at the same time the concentration of the diol, were determined. ECH rapidly disappeared from mouse blood, being no longer detectable 20 min after treatment. In contrast, 3-chloro-1,2-propanediol was measurable up to 5 h after dosage. No difference was observed in the kinetic and metabolic behavior of ECH after single and repeated doses (50 and 200 mg/kg/day for 7 days). When 3-chloro-1,2-propanediol was tested, neither glutathione depletion nor epoxide hydrolase inhibition (evaluated with both styrene-7,8-oxide and ECH as substrates) could be detected in mouse liver. Finally, no difference in ECH blood kinetics or metabolism were observed in experiments in which the compound was administered (200 mg/kg) intraperitoneally in water or orally as a solution in dimethyl sulfoxide.     

Modification by cis-diamminedichloroplatinum (II) of the radiation dose-response curve for intestinal crypt cells in mice

The effect of cis-diamminedichloroplatinum (II) (c-DDP) on the shape of the radiation dose-response curve for mouse duodenal crypt cells was investigated. A priming X-ray dose was followed 18 h later by graded test doses (single doses or five equal fractions at 3-h intervals) with or without c-DDP. Curves were fitted by a linear quadratic (LQ) relationship. The drug modified the dose-response curve by enhancing both the alpha and the beta terms. Repair kinetics were analyzed in split-dose experiments. c-DDP caused a minor, nonsignificant decrease in the rate of repair after irradiation. The survival ratio after split-dose irradiation, when the same X-ray doses were given, was actually slightly increased by the drug. This paradoxical effect can be explained by the fact that c-DDP mainly increased the beta term in the LQ relationship. There was no significant increase in crypt cell survival when split-drug doses were given alone at increasing intervals, suggesting no cellular repair after c-DDP treatment. The data are discussed in the light of the recently proposed "lethal and potentially lethal" (LPL) unified repair model of Curtis.     

Changes in X-ray sensitivity and glutathione content of human colon tumor cells after exposure to the differentiation-inducing agent sodium butyrate

Clone A human colon cancer cells were exposed to concentrations of sodium butyrate (NAB, 0-2 mM) for three passages in vitro, and responses to either graded single doses or split doses of 250 kVp X rays were determined. The survival data were fit to the single-hit, multitarget model of inactivation. For the graded single dose experiments, we found that NAB produced a decrease in the magnitude of the quasi-threshold (Dq) parameter after a concentration of about 0.9 mM was exceeded. Similarly, in split dose experiments, the amount of sublethal damage recovery (SLDR) was reduced in a concentration-dependent manner as shown by a decrease in the Dq parameter. However, the inhibition of SLDR occurred with no apparent threshold NAB concentration. NAB did not affect potentially lethal damage recovery. Paradoxically, increasing concentrations of NAB produced an exponential increase in the intracellular glutathione content, which could be blocked by exposure of the cells to buthionine sulfoximine (BSO). BSO treatment of NAB-adapted cells led to additional cell killing, again most noted by changes in the Dq parameter. We postulate that these responses are associated with NAB-induced changes in chromatin structure, particularly the association between DNA and nucleosomal histones H3 and H4.     

Mouse epidermal cell renewal after topical treatment with different concentrations of the epidermal peptide pyroGlu-Glu-Asp-Ser-GlyOH

Earlier work has shown that epidermal cells contain a peptide, pyroGlu-Glu-Asp-Ser-GlyOH, that induces a moderate but long-lasting inhibition of epidermal cell proliferation when given at low (picomol) doses ip in vivo and in vitro. In the present study, the epidermal pentapeptide was applied topically to the back skin of hairless mice at different concentrations and in a water-miscible cream. A single topical application of either high (0.25% wt/wt) or low (0.004% or 0.02% wt/wt) doses of the pentapeptide was followed by oscillations in epidermal DNA synthesis and G2-M cell flux (mitotic rate). In general, epidermal cell proliferation was inhibited during the first 10-day period after treatment with the two lower doses, while the highest concentration of pentapeptide (0.25%) stimulated epidermal cell proliferation. In spite of the effects on epidermal cell proliferation the size of the epidermal cell population in the treated area (number of nucleated cells and epidermal thickness) showed no corresponding alterations. The results could imply that the epidermal pentapeptide modifies epidermal cell proliferation and terminal differentiation in such a way that the two are balance with each other.     

Response of cultured IEC-17 normal rat intestinal epithelial cells to X radiation

The growth parameters and radiosensitivity of normal rat intestinal epithelial cells, IEC-17, were studied. The cells were cultured by standard methods and exposed to an array of doses (1-12 Gy) of 250 kVp X rays. The survival curves generated exhibited no initial shoulder and were bimodal. The Do of the first component was about 0.2 Gy and the second component. 5.0 Gy. The ability of this cell line to repair sublethal lesions was examined by fractionation studies; repair was completed within 60 min after the first dose. When Chinese hamster ovary (CHO) cells were grown under the same conditions used for the IEC-17 cells and then irradiated with single doses, a typical survival curve with a Do of 1.4 Gy was obtained. The survival curves obtained for the IEC-17 cell line are consistent with the response of a morphologically distinct single population containing two functionally separate types of cells.     

Protection by WR-3689 against gamma-ray-induced intestinal damage: comparative effect on clonogenic cell survival, mouse survival, and DNA damage

The aminophosphorothioate WR-3689 was characterized for its ability to protect mouse jejunal cells in vivo from single doses of X or gamma radiation. First, the effect of the drug on the survival of jejunal stem cells was examined using a clonogenic end point, the crypt microcolony assay. When WR-3689 was administered 30 min prior to whole-body irradiation, the number of surviving crypt cells was markedly increased at all doses of the drug, although protection began to level out at doses larger than 600 mg/kg. Protection was maximal when the drug was given 30 min before whole-body irradiation and declined rapidly with both shorter and longer intervals. Protection factors (PFs) were obtained by measuring survival curves for clonogenic crypt cells as a function of radiation dose; WR-3689 given 30 min before whole-body irradiation protected jejunum in the microcolony assay with a PF of 1.26 +/- 0.02, 1.50 +/- 0.10, and 1.65 +/- 0.10 at doses of 200, 400, and 800 mg/kg, respectively. Next, the effect of WR-3689 on the survival of jejunal stem cells was determined by assaying the survival of mice given X-ray doses to the whole abdomen in the range leading to death from the gastrointestinal syndrome. The PFs based on the LD50 values for 11-day survival were 1.31 +/- 0.05 (200 mg/kg) and 1.48 +/- 0.05 (400 mg/kg). Crypt-cell survival and animal survival were thus modified to a similar extent by this agent. Finally, the effect of WR-3689 on the induction of DNA single-strand breaks (SSBs) in jejunal cells was measured using an adaptation of the alkaline elution methodology. In mice treated with WR-3689 (400 or 800 mg/kg) 30 min prior to whole-body irradiation with 10 Gy there was no significant reduction in the number of DNA SSBs induced either in samples of the jejunum or in the cycling crypt cells, providing further evidence that there is no simple relationship between the modification of DNA SSBs and the survival of jejunal stem cells.     

Comparison of ES cell fate in sandwiched aggregates and co-cultured aggregates during blastocyst formation by monitored GFP expression

Markers and the means to detect them are required to monitor the fate of living cells. However, few suitable markers for living cells were known until a green fluorescent protein (GFP) was discovered. We have established mouse embryonic stem (ES) cell lines that express mutant GFP under the chicken beta-actin (CAG) promoter. Using these cell lines, we were able to follow the migration of ES cells during blastocyst formation both in sandwiching and coculture methods, even if only a single ES cell was used. Furthermore, the contribution of ES cells to the inner cell mass (ICM) was easily estimated at the blastocyst stage. We compared sandwiching with coculture aggregation relative to the contribution of the ES cell in the ICM, and the results indicated that there was no difference in the ratios of chimeric embryos having ICM contributed from cultured ES cells. Furthermore, an aggregated single ES cell was able to contribute three or four cells to the ICM at the blastocyst stage. Thus we conclude that one, instead of two, embryos is enough to make aggregation with ES cells, and a single ES cell attached to an embryo is enough to produce germline chimeras. Moreover, we could clearly observe single cell fate during blastocyst formation. This suggests that our established cell line can be used for monitoring single cell fate in vivo. In addition, we have shown that up to five doses of 30 sec of UV irradiation using GFP filters have no effect on the embryonic development.     

Intracellular calcium 'signatures' evoked by different agonists in isolated bovine aortic endothelial cells.

Agonist induced increases in intracellular free calcium, [Ca2+]i, were measured in single Fura-2 loaded bovine aortic endothelial (BAE) cells by dual wavelength microspectrofluorimetry. Low doses of ATP (less than 10 microM) induced complex changes in [Ca2+]i. These changes usually consisted of a large initial transient peak with subsequent fluctuations superimposed upon a maintained rise in [Ca2+]i. Higher doses of ATP (greater than 50 microM) produced much simpler biphasic increases in [Ca2+]i in individual cells. Acetylcholine and bradykinin also elicited increases in [Ca2+]i in single cells in confluent monolayers of endothelial cells. However, only acetylcholine produced complex fluctuations. High doses of acetylcholine evoked simple rises in [Ca2+]i similar to those seen with high doses of ATP. In contrast, bradykinin evoked relatively simple rises in [Ca2+]i at all doses used. These results indicate that the mechanisms responsible for generating agonist induced increases in [Ca2+]i in BAE cells are not homogeneous. ATP and acetylcholine produced more complex Ca2+ changes or 'signatures' in single confluent bovine aortic endothelial cells than bradykinin. All three agonists appeared to release Ca2+ from intracellular stores as well as stimulating Ca2+ influx. The possible mechanisms underlying these phenomena are discussed.     

Characteristics of the mechanism of action of complex copper (II) compounds with alpha-amino acids

There was no direct inhibition of DNA synthesis in ascites hepatoma 22A cells after intraperitoneal injection of single doses of copper (II) complexes with amino acids into tumor-bearing C3HA mice. Meanwhile cis-dichlorodiamine platinum (II) (DDP) as well as sarcolysine showed such inhibition. Copper (II) complexes with alpha-amino acids displayed as significant superoxide dismutase-like activity at concentrations corresponding to therapeutic doses of these compounds. The complexes of copper (II) combined with DDP give an additive antitumor effect in solid tumors of mice.     

Regulation of cell signaling dynamics by the protein kinase-scaffold Ste5

Cell differentiation requires the ability to detect and respond appropriately to a variety of extracellular signals. Here we investigate a differentiation switch induced by changes in the concentration of a single stimulus. Yeast cells exposed to high doses of mating pheromone undergo cell division arrest. Cells at intermediate doses become elongated and divide in the direction of a pheromone gradient (chemotropic growth). Either of the pheromone-responsive MAP kinases, Fus3 and Kss1, promotes cell elongation, but only Fus3 promotes chemotropic growth. Whereas Kss1 is activated rapidly and with a graded dose-response profile, Fus3 is activated slowly and exhibits a steeper dose-response relationship (ultrasensitivity). Fus3 activity requires the scaffold protein Ste5; when binding to Ste5 is abrogated, Fus3 behaves like Kss1, and the cells no longer respond to a gradient or mate efficiently with distant partners. We propose that scaffold proteins serve to modulate the temporal and dose-response behavior of the MAP kinase.