DNA synthesis rate and unlabeled cells with S-phase DNA content in P388 leukemic cells in vivo studied by flow cytometry and cell sorting

DNA synthesis kinetics of P388 leukemic cells growing in ascites form in BDF1 hybrid mice were investigated during the periods of exponential growth and growth restriction. Incorporation of tritiated thymidine, and in some instances tritiated uridine, was studied by autoradiography in cells sorted from S-phase fractions during DNA flow cytometry. During exponential growth continuous labeling with tritiated thymidine indicated a growth fraction of unity, whereas the growth fraction was about 30% during growth restriction. At this growth phase the majority of cells with S phase DNA content remained unlabeled after pulse labeling with tritiated thymidine or uridine, indicating that both the "salvage" and the "de novo" DNA synthesis pathways were blocked in most S-phase cells. After pulse labeling with tritiated thymidine the DNA synthesis rate pattern was investigated by sorting of consecutive fractions of cells throughout the S phase followed by quantitative autoradiography. With exception of a reduced rate in the middle of S phase, the DNA synthesis rate increased as the cells progressed through S phase during exponential growth. In contrast, the DNA synthesis rate pattern had a relative peak in the middle of S phase during growth restriction, which is otherwise characterized by a low mean DNA synthesis rate.     

Effect of N-trifluoroacetyladriamycin-14-valerate on [3H]thymidine uptake and DNA synthesis of human lymphoma cells

The effects of N-trifluoroacetyladriamycin-14-valerate on the uptake of [3H]thymidine and its incorporation into DNA of human P3HR-1 lymphoma cells were studied. In the absence of the drug, at 0 degrees C, [3H]thymidine was transported into the cells but not incorporated into DNA, as determined by both the trichloroacetic acid-soluble and -precipitable counts obtained with the cells. At 37 degrees C, [3H]thymidine was readily transported into the cells and incorporated into DNA. In the presence of the drug, both [3H]thymidine uptake (as shown by acid-soluble counts) and the amount of its incorporation into acid-precipitable materials were markedly reduced. However, the uptake of [3H]thymidine at 0 degrees C was found to be equally sensitive to drug inhibition as at 37 degrees C. The incorporation at 37 degrees C of [3H]thymidine into acid-precipitable materials of the cells, which had been prelabeled at 0 degrees C with [3H]thymidine, was found to be insensitive to inhibition by the drug. The in vitro activities of DNA polymerases alpha and beta purified from human P3HR-1 cells were also found not to be susceptible to inhibition. Nuclei purified from cells pretreated with the drug continued to synthesize DNA. The cytofluorograms of the cells treated with the drug indicated that the treated cells accumulated at the G2/M phase, whereas the S phase of the cells was not arrested. These results suggest that N-trifluoroacetyladriamycin-14-valerate inhibits [3H]thymidine uptake but not cellular DNA synthesis in human P3HR-1 lymphoma cells.     

Inhibition of DNA synthesis in adrenocortical cells by cytochalasin B

ACTH inhibits DNA synthesis in normal rat and mouse tumor Y-1 adrenocortical cells within the same concentration range that it stimulates steroidogenesis. These processes can be independently regulated as demonstrated by the divergent actions of cytochalasin B on these cells. In the normal cells, cytochalasin B does not increase steroidogenesis in serum-free or serum-containing media, and it decreases the stimulation produced by ACTH. In the absence of serum, the Y-1 cells respond in a similar way. However, in serum-containing media, cytochalasin B increases steroidogenesis in these cells and does not inhibit the response to ACTH. In both cell types, cytochalasin B inhibits [3H]thymidine incorporation into DNA by a mechanism different than that of ACTH. In the Y-1 cells, this inhibition is caused by a decreased uptake of [3H]thymidine into the cell, which probably reflects a decreased transport across the cell membrane. In the normal cells, cytochalasin B, like ACTH, does not affect [3H]thymidine transport, but it decreases DNA synthesis much more rapidly than does ACTH. This inhibition may be the result of the disruption of microfilaments by cytochalasinB, because our evidence indicates that it is not caused by a decrease in glucose uptake by the cells.     

Cytotherapy with naive rat umbilical cord matrix stem cells significantly attenuates growth of murine pancreatic cancer cells and increases survival in syngeneic mice

Background aimsPancreatic cancer, sometimes called a ‘silent killer’, is one of the most aggressive human malignancies, with a very poor prognosis. It is the fourth leading cause of cancer-related morbidity and mortality in the USA.MethodsA mouse peritoneal model was used to test the ability of unengineered rat umbilical cord matrix-derived stem cells (UCMSC) to control growth of pancreatic cancer. In vivo results were supported by various in vitro assays, such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), direct cell count, [³H]thymidine uptake and soft agar colony assays.ResultsCo-culture of rat UCMSC with PAN02 murine pancreatic carcinoma cells (UCMSC:PAN02, 1:6 and 1:3) caused G0/G1 arrest and significantly attenuated the proliferation of PAN02 tumor cells, as monitored by MTT assay, direct cell counts and [³H]thymidine uptake assay. Rat UCMSC also significantly reduced PAN02 colony size and number, as measured by soft agar colony assay. The in vivo mouse studies showed that rat UCMSC treatment significantly decreased the peritoneal PAN02 tumor burden 3 weeks after tumor transplantation and increased mouse survival time. Histologic study revealed that intraperitoneally administered rat UCMSC survived for at least 3 weeks, and the majority were found near or inside the tumor.ConclusionsThese results indicate that naive rat UCMSC alone remarkably attenuate the growth of pancreatic carcinoma cells in vitro and in a mouse peritoneal model. This implies that UCMSC could be a potential tool for targeted cytotherapy for pancreatic cancer.     

Cytochemical and ultrastructural studies of ribonucleoprotein containing structures in oocytes of Acheta domesticus

Summary Two distinct types of ribonucleoprotein containing structures are found in oocytes of the house cricket, Acheta domesticus, a large secondary or accessory nucleolus and many small primary nucleoli. The secondary nucleolus increases in size during oocyte development and is similar in appearance to the nucleolus of somatic cells. The primary nucleoli are intimately associated with a large, extrachromosomal DNA containing body. The DNA body is no longer visible in nuclei of late diplotene stage cells when the primary nucleoli are dispersed within the nucleoplasm. Both types of nucleoli contain cytochemically detectable RNA and acid protein, little or no DNA and basic protein, and particulate structures similar to but smaller than cytoplasmic ribosomes.The authors acknowledge the technical assistance of Miss Celeste Malinoski and Mrs. Marcia Andrews. This work was supported by a U.S.P.H.S. grant, number GM-16440-01 and grants number L-16 and J-1 from the Health Research Services Foundation.     

Flow cytometric measurement of cell cycle kinetics in rat Walker-256 carcinoma following in vivo and in vitro pulse labelling with bromodeoxyuridine.

Flow cytometric measurements of total DNA content, cell cycle distribution, and bromodeoxyuridine (BrdUrd) uptake were made in rat Walker-256 carcinoma cells. After both in vivo and in vitro pulse labelling with BrdUrd, Walker-256 tumor cells were stained with propidium iodide (PI) to estimate the total DNA content and a monoclonal antibody against BrdUrd to estimate the relative amount of cells in S phase. BrdUrd-labelled single cell suspensions were harvested at different time intervals to determine the movement of these cells within the cell cycle. To increase BrdUrd uptake, fluorodeoxyuridine (FDU), a thymidine antagonist, was also applied in vivo and in vitro. The results indicated exponential growth characteristics for this tumor between days 5 and 8 after implantation. Tumor doubling times, derived from changes in tumor volume in vivo and from the increase in cell number in vitro were similar. The mean time for DNA synthesis was estimated from the relative movement of BrdUrd-labelled cells towards G2. The percent of cells labelled with BrdUrd and the DNA synthesis time were similar regardless of the mode of BrdUrd administration. This study demonstrates that BrdUrd labelling of rat Walker-256 carcinoma cells in vitro yields kinetic estimates of tumor proliferation during exponential growth similar to those with the administration of BrdUrd in the intact tumor-bearing rat.     

Phospholipid metabolism in Ehrlich ascites tumor cells. I. Turnover rate of phosphatidylinositol.

1. Radioactive precursors, 32 PI, [1-14C]glycerol, and [1-14C]acetate, were individually injected into the peritoneal cavity of mice bearing Ehrlich ascites tumor, and the rates of incorporation into phospholipid fraction of Ehrlich ascites tumor cells were estimated. Although no distinct difference in specific activities was observed between phosphatidylinositol and other phospholipid classes as regards the incorporation of [1-14C]acetate of [1-14C]glycerol, a higher rate of incorporation of 32Pi into phosphatidylinositol was observed. The specific activity of phosphatidylinositol reached more than ten times that of phosphatidylcholine in the first hour. 2. The radioactivities incorporated into the phospholipids of Ehrlich ascites tumor cells and liver were estimated after simultaneous injection 32Pi and [2-3H]inositol. The incorporation of 32Pi into phosphatidylinositol of liver was similar in specific activity to those of other phospholipids. The ratio (3H/32Pi) of phosphatidylinositol only slightly in the ascites tumor cells, while an appreciable decrease of the ratio was observed in the liver during the first 3 hr. 3. These results suggest that phosphatidylinositol synthesis through pathways other than de novo synthesis is rapid in ascites tumor cells.     

Effect of microenvironment on functional activity of murine B-lymphocytes

In contrast to B-splenocytes, murine peritoneal B-cells do not produce or secrete immunoglobulins (Ig). Twenty-four hours after intraperitoneal transfer of splenocytes containing Ig-forming cells (IFC), IFC content in the peritoneal cavity of recipient mice decreases dramatically. This decrease does not depend on the migration of transferred IFC to the spleen (homing); splenectomy has no effect on this process. In order to check whether the lack of IFC in the peritoneal cavity is due to inhibition of synthesis or secretion of Ig, peritoneal cells (contained 4360 IFC/10⁶ cells) of CBA mice were intraperitoneally transferred to CBA/N mice after a 4-day preincubation in vitro. On the next day, ～30% of in vitro transferred IFC were detected in the peritoneal cavity of recipient mice, but on day 4 the IFC content in peritoneum returned to the background level. Repeated in vitro incubation of peritoneal cells of recipient mice restored the IFC number in cultured cells. It means that peritoneal microenvironment inhibits functional activity of murine B-lymphocytes.     

Effects of surface active agents on the susceptibility of Swiss mice to Candida albicans

Summary The LD50's ofCandida albicans combined with Plurafac B26, Mulsor 224, Pluronic L62, and Polyethylene Glycol 400 Mono Laurate injected intraperitoneally into female white Swiss mice were calculated by the method ofReed &Muench. Each of the previously mentioned surface-active agent —Candida albicans combinations had a lower LD50 than theCandida albicans control.Plurafac B26 was found to intially decrease the number of leukocytes in the peritoneal cavity and thus enhance the invasiveness ofCandida albicans.     

Activity of tumor-associated lymphoid cells at short intervals after administration of irradiated syngeneic and allogeneic tumor cells.

After a single intraperitoneal injection of irradiated tumor cells, host cells capable of responding against syngeneic tumors were detected in peritoneal exudates of mice. Although irradiation of the injected tumor prevented its overgrowth, it did not significantly alter the antigenicity of the tumor. Immunologic activities of tumor-associated host cells in the peritoneal cavity were continuously monitored, starting 48 hr after tumor administration. In vitro cell-mediated lysis of syngeneic tumors appeared as early as 3 days after irradiated tumor administration. In addition, peritoneal exudate cells from inoculated mice were capable of adoptively transferring immunity. Purification of these peritoneal exudate cells on nylon wool columns yielded a nonadherent Ig-negative lymphocyte fraction whose cytolysis was tumor-specific and T cell-associated. The macrophage-free lymphocyte fraction exhibited a higher in vitro activity against tumors than unpurified peritoneal exudates. This tumor-host system allowed the study of cells which directly interact with the tumor cells in vivo, starting shortly after tumor administration. The results reported in this paper show that tumor-associated lymphoid cells capable of mounting anti-tumor response in vivo and in vitro can be demonstrated as early as 3 days after tumor inoculation.     

Inhibition of mammalian DNA replication by dichlorobenzimidazole riboside

We have investigated the effect of the nucleoside analogue 5,6-dichloro-1-β- -ribofuranosyl-benzimidazole (DRB) on DNA synthesis in the L-929 line of mouse fibroblasts. Earlier studies have shown that this compound selectively inhibits the synthesis of a major fraction of nuclear heterogeneous RNA (hnRNA). At 60–75 μM, DRB decreases the rate of nuclear hnRNA synthesis by two-thirds and prevents the appearance in the cytoplasm of almost all poly(A)-containing messenger RNA (mRNA). At similar dose levels, DRB inhibits the overall rate of DNA synthesis as measured by thymidine incorporation by only 20% (after correction for decreased thymidine uptake into total cellular material). Analyses of density gradients of BUdR and [³H]thymidine-substituted DNA and of autoradiograms of extended fibers of [³H]thymidine-labeled DNA confirm this inhibition and suggest that DRB acts on DNA solely by reducing the rate of replication fork movement. The apparent size of replication units is unchanged by drug treatment. DRB also inhibits the transport of thymidine into cells. As determined by kinetic studies of thymidine uptake, transport is inhibited competitively by drug treatment. Several other aspects of thymidine metabolism are not affected by DRB. It does not alter the size of the total dTTP pool, and there is no breakdown of template DNA in DRB-treated cells.     

CYCLIC CHANGES IN THE CELL SURFACE : I. Change in Thymidine Transport and Its Inhibition by Cytochalasin B in Chinese Hamster Ovary Cells

Cytochalasin B (CB) shows a marked concentration-dependent inhibition of the incorporation of [³H]thymidine into Chinese hamster ovary cells. This inhibition was shown to result from an inhibition of thymidine uptake, not from an inhibition of DNA synthesis. Cells normally acquire the capacity to transport thymidine as they move from the G1 stage of the cell cycle into the S phase. If CB is added to cells while they are in G1, they do not acquire the ability to transport thymidine as they enter S. However, the addition of CB to cells that are already in S has no effect on their ability to transport thymidine. These results are discussed in terms of a model in which elements involved in thymidine transport enter the cell surface membrane as the cells move from G1 to S. It is proposed that CB prevents this structural transition by binding to the cell surface.     

Decreased Sensitivity to Hydroxyurea and to [3H]Thymidine Suicide In the Middle of the S Phase

Pluripotent haemopoietic stem cells (CFUs) move synchronously through the cell cycle in hydroxyurea-treated mice in a cohort 1–2 hr broad. Ten to fifteen hours after hydroxyurea they pass through S phase. DNA synthesis appears to be depressed 5–10 times when the cells are in the middle part of the S phase but does not seem to be completely interrupted. High concentrations of [³H]thymidine must be used for ‘suicide’ in order to achieve lethality for the cells with depressed DNA synthesis. At the time when DNA synthesis is depressed, the sensitivity of the cells to hydroxyurea also decreases. This may lead to a significant underestimation of the S phase fraction by the hydroxyurea method, because CFUs with low DNA synthesis rate are resistant to hydroxyurea although being in S phase.     

In vitro tumor cell killing by peritoneal macrophages from mitomycin C-treated rats

Summary The local cellular response induced by intraperitoneal injection of mitomycin C was examined in terms of cell-mediated cytotoxicity for tumor cells. An in vitro cytolysis assay involving ¹²⁵I-iododeoxyuridine-labeled tumor target cells revealed that treatment of normal ACI/N rats (200 g) with a single intraperitoneal injection of mitomycin C (50, 100, or 200 g) induced tumoricidal macrophages in the peritoneal cavity. The tumoricidal activity was dependent on the dose of mitomycin C injected and it was detectable as early as 1 day after the intraperitoneal injection of mitomycin C. In addition to the increased tumoricidal activity, the functional activities of the peritoneal macrophages were found to be increased with respect both to uptake of 2-deoxy-d-glucose and to phagocytosis of latex beads. Additional experiments excluded the possibility that the tumor cell cytolysis was the result of direct cytotoxicity by mitomycin C that might have been incorporated in the peritoneal macrophages or of nutrient depletion in the medium during the cytolysis assay. Furthermore, endotoxin contamination of the mitomycin C, which might have produced the activated macrophages, was not detected. The mechanism by which mitomycin C injected intraperitoneally induced the tumoricidal macrophages locally remains uncertain; however, it is possible also in clinical situations.     

转移及非转移肿瘤移植后615小鼠血液流变学变化的研究

血道高转移瘤株FC、淋巴合并血道高转移瘤株U14、淋巴道高转移瘤株H22、非转移瘤株P615分别接种于336只纯系近交615小鼠.不同时间取血并处死动物,进行组织学及血液流变学检查.将转移瘤发展过程分为潜伏期、侵袭期、转移早、中、晚期,非转移瘤发展过程分为潜优期、增殖期、囊腔形成期及中心坏死期.本实验结果显示,不同转移能力及途径肿瘤发展的不同时期血液流变学变化规律不同,因而表明肿瘤侵袭、转移与血液流变学变化之间存在互为因果的紧密关系.其临床诊断及治疗意义被讨论.     

转移及非转移肿瘤移植后615小鼠血液流变学变化的研究

血道高转移瘤株FC、淋巴合并血道高转移瘤株U14、淋巴道高转移瘤株H22、非转移瘤株P615分别接种于336只纯系近交615小鼠.不同时间取血并处死动物,进行组织学及血液流变学检查.将转移瘤发展过程分为潜伏期、侵袭期、转移早、中、晚期,非转移瘤发展过程分为潜优期、增殖期、囊腔形成期及中心坏死期.本实验结果显示,不同转移能力及途径肿瘤发展的不同时期血液流变学变化规律不同,因而表明肿瘤侵袭、转移与血液流变学变化之间存在互为因果的紧密关系.其临床诊断及治疗意义被讨论.     

Differential effects of ovarian steroids and triphenylethylene compounds on macromolecular uptake and thymidine incorporation in the mouse uterus

In the rodent uterus, estrogen elicits a biphasic response i.e. an early phase (Phase I) and a late phase (Phase II). Estradiol-17 beta (E2) and estriol (E3), as well as triphenylethylene (TPE) compounds, CI-628 and clomiphene citrate (CC), were used to characterize Phase I and Phase II responses in uterine preparation for implantation in the mouse. While uterine macromolecular uptake (vascular permeability), a Phase I response, was studied in progesterone (P4)-primed animals, uterine [3H]thymidine incorporation (DNA synthesis), a Phase II response, was investigated with and without P4-priming. In the P4-primed uterus, all compounds, except CC, significantly increased uterine macromolecular uptake as determined by interstitial tissue accumulation of [125I]bovine serum albumin [( 125I]BSA). DNA synthesis as determined by cellular incorporation of [3H]thymidine was modulated by P4, estrogens and TPE compounds in a cell-type specific and temporal manner. As a single injection and in the absence of P4, E2 induced [3H]thymidine incorporation in the luminal and glandular epithelium at 18 and 24 h. E3 was inferior to E2 in this response. On the other hand, treatment with P4 for 1 day or 4 days induced [3H]thymidine incorporation primarily in stromal cells. However, stromal cell incorporation was potentiated when P4 treatment was combined with estrogens or TPE compounds. These results reveal the relative importance of Phase I and cell-type specific Phase II responses in uterine preparation for implantation.     

Inhibition of macrophage DNA synthesis by immunomodulators. II. Characterization of the suppression by muramyl dipeptide or lipopolysaccharide [3H]thymidine incorporation into macrophages

Guinea pig peritoneal exudate macrophages actively incorporated [3H]thymidine into trichloroacetic acid-insoluble fraction in vitro. The incorporation of [3H]thymidine was almost completely inhibited by aphidicolin, an inhibitor of DNA polymerase alpha and an autoradiograph showed heavy labeling in nuclei of 15% of macrophage populations. These results indicate that the observed thymidine incorporation was due to a nuclear DNA synthesis. The [3H]thymidine incorporation was markedly suppressed when macrophages were activated by immunoadjuvants such as muramyl dipeptide (MDP) or bacterial lipopolysaccharide (LPS). The suppression of [3H]thymidine incorporation by MDP was neither due to the decrease in thymidine transport through the cell membrane, nor due to dilution by newly synthesized "cold" thymidine. An autoradiograph revealed that MDP markedly decreased the number of macrophages the nuclei of which were labeled by [3H]thymidine. These results suggest that the suppression of [3H]thymidine incorporation by the immunoadjuvants reflects a true inhibition of DNA synthesis. The inhibition of DNA synthesis by MDP was also observed in vivo. Further, it was strongly suggested that the inhibition was not caused by some mediators, such as prostaglandin E2, released from macrophages stimulated by the immunoadjuvants but caused by a direct triggering of the adjuvants at least at the early stage of activation. Cyclic AMP appears to be involved in the inhibitory reaction.