Changes of the surface proteolytic activity in synchronized Ehrlich ascites tumor cells grown in vivo

Summary Changes of the cell surface proteolytic activity during the cell cycle in vitro were reported. Using an easy assay, with casein as a substrate, the proteolytic activity on the surface of Ehrlich ascites tumor (EAT) cells grown in vivo was determined. The cleavage of casein incubated with EAT cells increased linearly for 20 min and permitted reproducible enzyme activity determinations. If the proliferation of exponentially growing EAT cells was partially synchronized by an intraperitoneal bleomycin injection, a significant increase of the surface enzymatic activity was observed in cells with an increased DNA content. This finding supports the results obtained with transformed cells in vitro, indicating that elevated proteolytic surface activity occurs in the late synthesis phase and prior to mitosis. However, the observed effect may also be due to changes of gene expression caused by bleomycin.Abbreviation used EAT Ehrlich ascites tumor     

EVIDENCE FOR CONTROL OF THE RATE OF NUCLEAR DNA SYNTHESIS BY THE NUCLEAR MEMBRANE IN EUKARYOTIC CELLS

Rates of DNA synthesis in root tip cells of diploid and autotetraploid snapdragon (Antirrhinum majus) seedlings and in diploid and tetraploid (mononucleate and bidiploid-nucleate) regenerating mouse-liver cells have been studied. The increase in this rate in the tetraploid cells is closely correlated with the increase in nuclear surface area, but not with the nuclear volume; this suggests a possible control of the rate of DNA synthesis by the nuclear membrane.     

Biosynthesis of ferritin subunits from different cell lines of HL-60 human promyelocytic leukaemia cells and the release of acidic isoferritin-inhibitory activity against normal granulocyte-macrophage progenitor cells

Biosynthesis of acidic isoferritins was investigated in human promyelocytic HL-60 cells, characterized by diploid (2C), tetraploid (4C) and mixed diploid–tetraploid (2C–4C) DNA cell lines. The three cell lines were studied for the biosynthesis of ferritin and its subunits and for the release of acidic isoferritin-inhibitory activity against normal CFU-GM before and after addition of DMSO. While the tetraploid and mixed diploid–tetraploid cell lines synthesized more H- ( M _r= 21) than L-subunits ( M _r= 19) after induction, the tetraploid line synthesized more H-subunit before and after induction, compared to the diploid line. The release of acidic isoferritin-inhibitory activity was greater before than after induction in both cell lines, but the tetraploid cell line released more acidic isoferritin-inhibitory activity consistent with its greater production of M _r= 21 subunit. However, after induction no inhibitory activity could be detected from the diploid cells and much less activity was detected with the tetraploid cells, suggesting that differentiation caused a decrease in production of acidic isoferritin-inhibitory activity.     

Giant cell formation in cultured L5178Y lymphoblasts induced by hydroxyurea treatment

The conditions leading to hydroxyurea-induced abnormal cell enlargement (giant cell formation) were studied in L5178Y lymphoblasts in the culture. Exposure for only 1 hour to a concentration of 1 mM hydroxyurea (HU) was sufficient to produce an abnormal enlargement of about 25 percent of the cells. The maximal proportion of giant cells reached the value of about 50 percent after treatment with 1 mM HU for 5 hours and was not changed by a further prolongation of exposure time and/or by increasing the concentration of HU to 10 mM. Comparison of cell populations with various proportions of giant cells as regards their ability to reproduce, DNA synthesis and persistence in the culture suggested that at least some part of giant cells lost the ability to divide, their DNA synthesis was markedly suppressed or retarded and more than 50 percent of them disappeared earlier than 48 hours after the termination of HU exposure. It is concluded that at least at certain HU concentrations and in suitable stage of target cells the response of cells to short HU treatment can resemble that produced by X-rays or other DNA damaging agents.     

Polyploidy in the pancreas of the normal and diabetic mutant mouse

Summary The DNA content of endocrine and exocrine pancreatic nuclei of normal C57BL/KsJ and diabetic mutant C57BL/Ks-db/db mice was measured by Feulgen microdensitometry. The exocrine and endocrine pancreatic nuclei of the 4-week-old normal, 12-week-old normal, and 4-week-old (prehyper-glycaemic) diabetic mutant mice contained diploid and tetraploid cells, while the 12-week-old (established hyperglycaemic) mutant contained diploid, tetraploid, and octaploid nuclei. The polyploidy in the endocrine pancreas of all these mice was confined to the B-cells, while the A-cells were always diploid.     

Cytotoxicity of adriamycin,idarubicin, and vincristine in acute myeloid leukemia: Chemosensitization by verapamil in relation to P-glycoprotein expression

Summary A 4-day colorimetric tetrazolium dye (MTT) assay was used to assess the cytotoxicity of adriamycin (ADM), vincristine (VCR), and idarubicin (IDA) in blasts isolated from 37 patients with newly diagnosed and pretreated acute myeloid leukemia (AML). The effect of verapamil (VRP) as a chemosensitizer was studied in relation to the expression of the membrane efflux pump P-glycoprotein (PGP) as determined by a semiquantitative flow-cytometric procedure. A slight positive correlation was found between the fraction of cells expressing PGP and the ID₅₀ values for ADM and VCR, but not between cellular PGP content and sensitivity to IDA. The overall data showed no significant sensitization effect of VRP. However, in specimens with more than 10% cells expressing PGP, 2M VRP sensitized cells to ADM and VCR significantly. The median of sensitization ratios (SRs), i.e., the ratios of cytotoxic drug ID₅₀ in the absence/presence of VRP, were 1.89 and 2.0, respectively. No sensitizing effect of VRP on the cytotoxicity of IDA was observed. Related to the clinical status, the median fraction of PGP-positive blasts was elevated fourfold in pretreated patients (n=16) in comparison to patients with de novo AML (n=19). No differences in ID₅₀ values were observed between newly diagnosed and pretreated patients. However, SRs for ADM and VCR were higher in samples of pretreated patients compared with de novo AML. PGP-mediated cellular drug resistance may thus be circumvented in leukemic blasts by application of chemosensitizers or, potentially, alternative anthracyclines.     

Uterine deoxyribonucleic acid synthesis during preimplantation in precursors of stromal cell differentiation during decidualization

During early pseudopregnancy, DNA synthesis and mitosis in the uterine endometrial stroma precede the development of uterine sensitivity to deciduogenic stimuli. Progesterone redirects the effects of estradiol on endometrial DNA synthesis from the luminal epithelium to the stroma. To determine the time and hormonal control of preimplantation endometrial DNA synthesis, uterine cells were labeled with [3H] thymidine at specific times during early pseudopregnancy or after progestin and estrogen treatment of ovariectomized rats. The fate of these labeled cells after their decidualization was examined by separation of prelabeled deciduomal cells by velocity sedimentation at unit gravity, which separates cells by size. Stromal cells that synthesized DNA during early pseudopregnancy or in response to hormone treatment later differentiated into deciduomal cells. Rates of DNA synthesis increased on days 4 and 5 of pseudopregnancy, with greater incorporation occurring on day 4 in cells that differentiated into polyploid deciduomal cells. In ovariectomized rats, medroxyprogresterone acetate treatment for 15 h increased DNA synthesis in stromal cells that differentiated into diploid and tetraploid deciduomal cells. DNA synthesis increased further at 30 h before returning to basal levels at 48 h. After progestin pretreatment, estradiol treatment increased stromal DNA synthesis again with greater incorporation in cells differentiating into polyploid deciduomal cells. These data indicate that during early pseudopregnancy, both progesterone and estradiol control the DNA synthesis of endometrial stromal cells as a means of reprogramming these cells for the later growth and differentiation of decidualization.     

Modification by caffeine of acute cytotoxic response of cultured L5178Y cells to hydroxyurea treatment

The effect of caffeine (CAF) on acute cytotoxic response of L5178Y lymphoblasts to hydroxyurea (HU) treatment was studied. The following events were examined: abnormal cell enlargement (giant cell formation), the rate of recovery of cell reproduction and DNA synthesis after releasing the cells from the HU blockage, parental DNA breakage and cell death. The presence of CAF at nontoxic concentration prevented giant cell formation, enhanced cell growth inhibition and cell killing. The effect of CAF was variable, dependent on the duration of exposure to HU and the time of exposure to CAF. To obtain maximal effect, the continuous presence of CAF during HU treatment and posttreatment time was necessary. Hydroxyapatite chromatography assay of single strand (ss) and double strand (ds) fractions in parental DNA and the measurement of the rate of post-treatment recovery of DNA synthesis indicated that CAF enhanced HU-induced DNA lesions. It is concluded that the results give further evidence that even short HU treatment can damage not only newly formed but also parental DNA. The lesions are normally, at least partly repaired and can be expressed under the conditions of DNA repair inhibition.     

DNA-synthesis and polyploidization of chicken heart muscle cells in mass cultures

Heart muscle cells from 9-day-old chick embryos were grown in vitro in three different media. We investigated DNA synthesis and proliferation and the periodic acid Schiff reaction (PAS) for identification of cardiac muscle cells (myocytes). No increase of the number of myocytes was observed, whereas non-muscle cells showed substantial proliferation. However, autoradiographic studies of cultures incubated for up to 5 days with [³H]thymidine revealed on average more than 50% of the myocytes synthesized DNA. Pulse labelling studies (2 h with [³H]thymidine) demonstrated a maximum of DNA synthesizing muscle cells (ca 40%) at day 3 of incubation.The DNA content of mononucleated myocytes measured by cytofluorometry showed a shift from a diploid to a tetraploid state, whereas the non-muscle cells behaved like a proliferating population.By comparison heart cells from 5-day-old embryos showed a slight increase in the number of myocytes (ca 20%); however, tetraploidization was also regularly observed.     

Decreased acquisition of glucocorticoid resistance in tetraploid human lymphoid cells

Resistance to dexamethasone (1 microM) was measured in glucocorticoid-sensitive diploid and tetraploid clones of the human leukaemic cell line CCRF-CEM (clone C7) during continuous culture and after X-ray or chemical mutagenesis. In continuous culture resistant diploid cells accumulated at a rate of about one cell per 10(5) divisions, while the rate for tetraploid cells was less than one per 10(7) divisions. Chemical and X-ray mutagenesis caused a marked increase in the number of resistant diploid cells but had very little effect on tetraploid cells. These results are consistent with a mutational basis for the acquisition of the glucocorticoid-resistant phenotype in human lymphoid cells.     

Pairwise loss of mitotic ability by human diploid fibroblasts

Non-transformed human diploid fibroblasts, like WI-38, cannot be maintained as permanent cell lines. With successive divisions in culture, the cells lose the ability to synthesize DNA. This loss may be due to inheritance, to a shared environment, or to interaction among offspring. We studied the pattern of DNA synthesis in WI-38 daughter pairs. A synchronized population in early prophase was seeded in cloning medium containing tritiated thymidine. Radioautographs were made after three days of incubation. We found that the loss of ability to undergo DNA synthesis was correlated between the members of daughter pairs. Daughter pairs in which one but not both of the cells had incorporated the labelled thymidine were significantly less frequent than if the loss of ability to synthesize DNA were to occur at random. The spatial pattern of loss is consistent with the release of a diffusible inhibitor of DNA synthesis by each newly-formed cell. Daughter pairs in which one or both of the cells incorporate thymidine occur with increasing frequency as the inter-nuclear distance between pair members increases. Interaction between daughter cells is responsible in part for the finite lifespan of human diploid fibroblasts.     

Suppression of the first stage of phorbol 12-tetradecanoate 13-acetate-effected tumor promotion in mouse skin by nontoxic inhibition of DNA synthesis.

In order to evaluate the significance of epidermal cell proliferation for the first stage of skin tumor promotion, the effect of hydroxyurea (HU), an inhibitor of DNA synthesis, on tumor formation was studied. Mice initiated with 7,12-dimethylbenz[a]anthracene received a single dose of phorbol 12-myristate 13-acetate (PMA) in stage I of promotion, followed by twice weekly application of the irritant skin mitogen phorbol 12-retinoate 13-acetate in stage II. A single dose of HU given intraperitoneally at different times before or after treatment with PMA was found to interfere with tumor formation, exhibiting an almost complete inhibition if administered 18 hr after PMA--i.e., at the time of maximal DNA synthesis. The inhibition of tumor formation by HU in the two-stage promotion experiment did not prevent a subsequent promotion of cells by repetitive PMA treatment. This indicates that the inhibitory effect of HU was due neither to cytotoxicity (killing of initiated cells) nor to an interference with initiation. The data indicate that epidermal DNA synthesis is obligatory for PMA-induced first-stage promotion. The causal relationship between both events remains to be established.     

DNA ploidy and protein synthesis in squamous cell carcinoma cell lines of the head and neck

Aneuploidy, as abnormal nuclear DNA content, is considered almost positive evidence of malignancy. In this study three diploid and three aneuploid squamous cell carcinoma (SCC) cell lines were examined for DNA content by flow cytometry. The DNA indices of the SCC cell lines were found to range from 1.0 to 2.1. The mitotic activity of the diploid cell lines was 1.6 times higher and the cells were smaller than aneuploid cells. To find a molecular basis for these differences, the pattern of the de-novo synthesized proteins was analyzed by means of [³⁵S]methionine incorporation, electrophoresis, and autoradiography. In all aneuploid SCC cell lines tested in this experiment, the increase of nuclear DNA content is associated with the synthesis of a novel protein with a molecular mass of approximate 55 kDa as well as with altered synthesis rates of two preexisting proteins (50 kDa and 100 kDa). For determination of the amino acid uptake in diploid and aneuploid cells, the accumulation of [³⁵S]methionine was measured as a function of time by liquid scintillation counting. No significant difference was found in the uptake rate between diploid and aneuploid cells with the same protein content. However, discrepancies were revealed when equal numbers of cells with different DNA index were used, suggesting, that protein turnover is different in diploid and aneuploid SCC cells.Abbreviations SSC squmous cell carcinoma - PBS phosphate-buffered saline This research was in part supported by the Schleswig-Holsteinische Krebsgesellschaft     

Stage-specific binding of inhibin and activin to subpopulations of rat germ cells.

Flow cytometry was used to separate and identify Sertoli and germ cell populations in primary rat testicular cultures derived from animals of different ages on the basis of cell size and DNA and lipid content. Multiparameter fluorescent evaluation of each cell preparation resulted in the assignment of specific staining patterns to Sertoli cells (diploid, high lipid content), spermatogonia (diploid, low lipid content), spermatocytes (large, tetraploid, high lipid content), and round spermatids (haploid, low lipid content). Each field was separately analyzed for inhibin and activin binding. Fluorescein isothiocyanate-conjugated activin bound with greatest intensity to spermatogonia, with little binding to leptotene or zygotene spermatocytes. Fluorescein isothiocyanate-conjugated inhibin bound to all stages of germ cells tested. Cross-competition data indicate that at least two and probably three distinct receptors exist for these peptides.     

Hydroxyurea enhances 3′-azido-3′-deoxythymidine (AZT) cytotoxicity in human chronic myeloid leukemia models*

Abstract: In this report we have evaluated the cytotoxic activity of 3′-azido-3′-deoxythymidine (AZT) used in combination with hydroxyurea (HU), an agent which disrupts de novo thymidylate synthesis. In 2 chronic myeloid leukemia (CML) cell lines, K.562 and RWLeu4, the IC50 of AZT was 8 (μmol/l and 28 μmol/l respectively, after a 5-day exposure, and the IC50 of HU was 80 μmol/l and 70 μmol/l respectively. In the presence of various concentrations of HU (1 μmol/l-100 μmol/l) the IC50 of AZT in both cell lines was significantly reduced and subsequent isobologram analysis revealed synergistic activity. Similarly, analysis of [3H]AZT incorporation into the DNA fraction of these cells indicated that exposure to AZT + HU resulted in an increased incorporation of AZT into DNA when compared to incubation in AZT alone. Biochemically, this effect appeared to be related to a decrease in dTTP pools caused by HU. The combination AZT + HU has also been demonstrated to exert a synergistic effect in inhibiting colony growth of bone marrow granulocyte-macrophage progenitors (CFU-GM) from patients affected by Ph1⁺ CML in chronic phase. These results are promising in view of a possible in vivo utilization of this drug combination.     

The rate of appearance of thyroid hormone nuclear receptor is increased during deoxyribose nucleic acid synthesis in GC cells: analysis of thymidine-treated GC cells using dense amino acid labeling

The growth rate of GH-producing rat pituitary tumor cells (GC cells) is dependent on thyroid hormone. Previous studies have shown that GC cells can be partially synchronized in the DNA synthesis phase (S-phase) of the cell cycle by a 25-h incubation with 2 mM thymidine. Measurements in GC cells partially synchronized in S-phase showed a significant increase in cellular thyroid hormone nuclear receptor appearance and disappearance in thymidine-treated GC cells using dense amino acid labeling and subsequent sucrose gradient ultracentrifugation. In comparison with GC cells in asynchronous culture, thymidine treatment causes a steady state increase in thyroid hormone nuclear receptors which results from an increase in the receptor appearance rate without an effect on the disappearance rate. Thus, position in the cell cycle appears to be another factor that influences the appearance rate of nuclear thyroid hormone receptors.     

The ultrastructure of polyploid B-cells in the islets of normal mice

Summary Light and electron microscopic studies of diploid, tetraploid and octaploid B-cells in the islets of normal C57BL/KsJ mice revealed that polyploid cells were characterized by a wider range of granulated states than diploid B-cells. The maximum granule densities were similar for polyploid and diploid cells; however, some polyploid cells were almost devoid of granules, while the least granulated diploid cells contained intermediate granule densities. The tetraploid cells also appeared to be characterized by an increased mitochondrial stage which suggests compensation for the greater degree of degranulation. These observations were confirmed by morphometric analysis. Two interpretations of the apparent polyploidy are discussed; that polyploid B-cells may be more responsive to insulin releasing stimuli than diploid B-cells and that tetraploid cells may only be diploid cells in the G₂ phase of the mitotic cycle.     

替莫唑胺对人胶质瘤细胞系U251细胞的增殖影响

目的探讨替莫唑胺（TMZ）对人胶质瘤细胞系U251细胞的杀伤作用及其作用机理。方法将人胶质瘤细胞系U251细胞分成空白对照组、TMZ组和常用化疗药物组,TMZ组下设10、25、50、100、200、400μmol／L组,常用化疗药物组（ADM、MTX、VCR、DDP）各药物浓度均为其IC50值。各组分别于作用后24、48、72h观察细胞形态学改变,用MTT比色法检测细胞活力,流式细胞仪检测细胞周期和凋亡率。结果①TMZ组各浓度对U251细胞的抑制率分别为4．23％、7．43％、31．63％、64．53％、82．18％、86．15％,呈良好的时间—剂量效应关系,其1％为81μmol,初始抑制浓度为50μmol;②ADM、MTX、VCR、DDP对U251细胞的抑制率分别为39．27％．44．59％、54．56％、55．28％,VCR、DDP两药的抑制作用明显优于ADM、MTX（P均〈0．01）,与TMZ的杀伤作用相当（P〉0．05）;③流式细胞仪检测显示,U251细胞经TMZ作用后出现G2／M期阻滞,但促凋亡作用不强。结论TMZ对胶质瘤细胞有明显的杀伤作用,并呈时间和剂量依赖性;TMZ可使胶质瘤细胞阻滞于G2／M期,但其促凋亡作用不强;替奠唑胺对U251细胞的杀伤作用优于其他4种常用化疗药物。