Cells In Different Phases Research Articles

Determination of the Effects of Camptothecin on Cell Cycling and Apoptosis in Human Leukaemic Cell Lines.

INTRODUCTION. The measurement of ATP is a recognised method for determination of proliferation or the effect of toxic environments on the cell. More recently, the relationship between ATP and ADP has been shown to be an important factor in determining the apoptotic or necrotic state of the cell (1). The ratio of ADP to ATP shows significant correlation with the degree of apoptosis, as measured by the estimation of the sub-G0 by propidium iodide staining of the nuclei and flow cytometry. Camptothecin is a topoisomerase inhibitor and as such, has effects on the cell cycle prior to inducing apoptosis. This compound has also been used as a chemotherapeutic agent in the treatment of a number of malignancies including leukemias. We have investigated these effects in a number of human leukaemic cell lines, concentrating on U937 and HL-60 cells, together with blast cells isolated from the peripheral blood of patients with acute myeloid leukemia. METHOD. HL-60 cells were incubated for 4 hours, while with the U937 cells a 24-hour incubation was required for induction of apoptosis, with increasing concentrations of camptothecin. The AML blast cells, from 10 patients were incubated with increasing concentrations of camptothecin, Ara-C and etoposide for 4 to 24 hours. The extent of apoptosis was shown by measurement of the ratio of ADP:ATP using the ApoGlow Kit (LumiTech (UK) Ltd). The presence of apoptotic events within the cell population was confirmed using Annexin V, JC-1, and propidium iodide (PI) staining and flow cytometry. The PI staining was also used to determine the relative percentage of cells in different phases of the cell cycle. RESULTS. The HL-60 and U-937 cells showed a concentration dependent effect on apoptosis induction with concentrations of camptothecin from 100 to 500nM. At the highest concentration of camptothecin used (1000nM) there was a significant reduction (n=7, p<0.0001) in the ratios compared with the 500nM sample. Comparison with the PI data revealed emergence of the G0/G1 peak showing that a subpopulation of cells was beginning to cycle again. With HL-60 cells at 1000nM of the drug, there was an increase in the percentage viable cells as shown by exclusion of PI, this correlated with a reduction in the level of ADP relative to ATP with the emergence of a resistant viable population. Of the AML cells investigated there was a marked difference in the response of the patient cells to the different drugs investigated, with respect to induction of apoptosis and drug resistance. DISCUSSION. The U937 cells displayed variations in responses according to the concentration of camptothecin used. The cells arresting in S-phase at low concentrations, could

Desynchronization Rate in Cell Populations: Mathematical Modeling and Experimental Data

We characterize the kinetics of two cancer cell lines: IGROV1 (ovarian carcinoma) and MOLT4 (leukemia). By means of flow cytometry, we selected two populations from exponentially growing in vitro cell lines, depending on the cells' DNA synthesis activity during a preceding labeling period. For these populations we determined the time course of the percentages of cells in different phases of the cycles, sampling every 3 hr for 60 hr. Initially, semi-synchronous populations quickly converged to a stable age distribution, which is typical of the cell line (at equilibrium); this desynchronization reflects the intercell variability in cell cycle duration. By matching these experimental observations to mathematical modelling, we related the convergence rate toward the asymptotic distribution (R) and the period of the phase-percentage oscillations (T), to the mean cell cycle duration and its coefficient of variation. We give two formulas involving the above-mentioned parameters. Since T and R can be drawn by fitting our data to an asymptotic formula obtained from the model, we can estimate the other two kinetic parameters. IGROV1 cells have a shorter mean cell cycle time, but higher intercell variability than the leukemia line, which takes longer to lose synchrony.

Cell Cycle Analysis and Synchronization of the XenopusCell Line XL2

We have determined the length of the cell cycle and its different phases in a permanent Xenopustadpole cell line, XL2. Following BrdU labeling, the total length of the cell cycle was estimated as 28 h. The different phases of the cell cycle, G 1, S, G 2, and M were, respectively, 14 h, 10 h 45 min, 2 h 30 min, and 54 min. Knowing these parameters, we were able to develop methods that selectively enrich cells in different phases of the cycle. Treatment with aphidicolin resulted in a S phase block in which more than 85% of the cells showed S phase chromosomes. Almost 60% of the cells were arrested in mitosis after a double block with aphidicolin/nocodazole or aphidicolin/ALLN (acetyl-leucyl-leucyl-norleucinal) treatment. This synchronization protocol will greatly facilitate studies of biochemical events associated with specific gene regulation through the cell cycle. Our synchronization protocol does not disturb cell metabolism as the expression of cyclin B 2during the cell cycle is in agreement with the results obtained with mammalian cells.

Green tea constituent epigallocatechin-3-gallate and induction of apoptosis and cell cycle arrest in human carcinoma cells.

The polyphenolic compounds present in green tea show cancer chemopreventive effects in many animal tumor models. Epidemiologic studies have also suggested that green tea consumption might be effective in the prevention of certain human cancers. We investigated the effect of green tea polyphenols and the major constituent, epigallocatechin-3-gallate, on the induction of apoptosis (programmed cell death) and regulation of cell cycle in human and mouse carcinoma cells. Human epidermoid carcinoma cells (cell line A431), human carcinoma keratinocyte (cell line HaCaT), human prostate carcinoma cells (cell line DU145), mouse lymphoma cells (cell line L5178Y), and normal human epidermal keratinocytes (NHEKs) were used. Apoptosis was assessed by 1) the formation of internucleosomal DNA fragments by agarose gel electrophoresis, 2) confocal microscopy, and 3) flow cytometry after tagging the DNA fragments by fluorescence label. The distribution of cells in different phases of the cell cycle was analyzed by flow cytometry. Treatment of A431 cells with green tea polyphenols and its components, epigallocatechin-3-gallate, epigallocatechin, and epicatechin-3-gallate, resulted in the formation of internucleosomal DNA fragments, characteristic of apoptosis. Treatment with epigallocatechin-3-gallate also resulted in apoptosis in HaCaT, L5178Y, and DU145 cells, but not in NHEK. Confocal microscopy and flow cytometry confirmed the findings. The DNA cell cycle analysis showed that in A431 cells, epigallocatechin-3-gallate treatment resulted in arrest in the G0-G1 phase of the cell cycle and a dose-dependent apoptosis. Green tea may protect against cancer by causing cell cycle arrest and inducing apoptosis. It needs to be evaluated in human trials.

Expression of corticotropin-releasing hormone and its R1 receptor in human endometrial stromal cells.

Corticotropin-releasing hormone (CRH) is a hypothalamic neuropeptide that has been identified also in several peripheral tissues, including organs of the reproductive system. In man, CRH is synthesized and released by the gonads, the placenta, maternal decidua, and the epithelial endometrium. So far, CRH has been demonstrated in endometrial stromal cells only after decidualization. The aim of this study was to seek evidence of the production and secretion of CRH by endometrial stromal cells in different phases of the menstrual cycle and to look for gene expression of the recently identified CRH receptor R1. Total RNA was extracted from stromal cells monolayers established from endometrial samples collected during both proliferative and secretive phases. After reverse transcription, polymerase chain reaction (PCR) amplification was carried out using primers specific to CRH and to CRH receptor R1, resulting in the expected bands, respectively 233 bp for CRH and 274 bp for CRH-R1. The identity of the obtained CRH PCR product was confirmed by restriction enzyme analysis and by Southern blotting. Purification by high performance liquid chromatography (HPLC) of stromal cell culture medium revealed a major peak of CRH immunoreactivity coeluting with the standard CRH(1-41), thus indicating the secretion of the mature peptide. Our study demonstrates the synthesis and secretion of CRH by endometrial stromal cells at all phases of the menstrual cycle. We also demonstrate the expression of the CRH receptor R1 gene. It can be hypothesized that CRH contributes via autocrine/paracrine mechanisms to endometrial physiology.

Cytomixis in Brassica napus var. oleifera and Brassica campestris var. oleifera (Brassicaceae).

During an investigation of the meiotic behavior of 11 canola cultivars (Brassica campestris and Brassica napus.), eight were found to present cytomixis between sporocytes. The percentage of cells with cytomixis was low, ranging from 0.06 to 2.55%. In two cultivars, cytoplasmic bridges without chromatin transfer between cells were higher than cytomixis. All meiotic phases from pachytene to tetrads were affected. Chromosome transfer was observed between cells in the same phase of division and also between cells in different phases of division. The amount of nuclear material transferred from one cell to the other was variable, involving, in some cases, the entire chromosome complement.

Effect of atriopeptides on proliferative processes in albino rat myocardium at the early stage of postnatal ontogenesis

The effect of atriopeptide AP-II and its 6-amino-acid acyclic fragment (7–12) AP-H-6-OH on proliferative processes in the myocardium of 5-day-old albino rats is examined 24 h after a single administration (200 nmol/kg intraperitoneally) by autoradiography with3H-thymidine and by analysis of mitotic regime. A significant decrease in the amount of DNA-synthesizing cells in the right atrium, right ventricle, and interventricular septum is recorded after administration of AP-II. Changes in DNA synthesis are attended by significant changes in the amount of cells in different phases of mitosis, indicating a decrease in the number of mitotic cells and a metaphasal delay. Administration of AP-H-6-OH causes no significant changes in DNA synthesis in the myocardium at the early stage of postnatal ontogenesis.

Behavior of Cryopreserved Endothelial Cells in Different Phases: Their Application in the Seeding of Vascular Prostheses

Various methods of cryopreservation of human endothelial cells (EC) were studied to determine their viability and behavior when seeded onto vascular prostheses made of polytetrafluoroethylene (PTFE). Three different protocols were used: (1) cyropreservation of whole umbilical vein, (2) cyropreservation of freshly extracted umbilical EC in suspension, and (3) cryopreservation of EC derived from a first subculture. Fresh EC and EC from a first subculture were used as controls. The viability and growth of these cells in culture media were studied, and basal prostacyclin levels were determined. The cells were assessed morphologically after they were seeded onto PTFE discs. Our results showed that the cryopreservation method that maintained the greatest viability was that in which previously cultured EC were used. Basal prostacyclin levels were significantly different following cyropreservation. However, when these cells were seeded onto PTFE discs their behavior was similar to that of fresh EC.

Preferential repair of the transcribed DNA strand in the dihydrofolate reductase gene throughout the cell cycle in UV-irradiated human cells

We examined repair of UV-induced cyclobutane pyrimidine dimers (CPD) in each strand of the expressed dihydrofolate reductase gene in human cells in different phases of the cell cycle: G1, early S, middle S, late S, and G2/M. After 4 h of incubation, repair of the transcribed strand was substantially more efficient than repair of the non-transcribed strand in all phases. Furthermore, we observed no remarkable cell cycle-dependent differences in either the initial lesion frequency or the efficiency of repair of the transcribed strand. We conclude that transcription coupled repair operates generally and with high efficiency throughout the cell cycle.

Synergistic Growth Inhibitory and Differentiating Effects of Trimidox and Tiazofurin in Human Promyelocytic Leukemia HL-60 Cells

Increased ribonucleotide reductase (RR) activity has been linked with malignant transformation and tumor cell growth. Therefore, this enzyme is considered to be an excellent target for cancer chemotherapy. We have examined the effects of a newly patented RR inhibitor, trimidox (3,4,5-trihydroxybenzohydroxamidoxime). Trimidox inhibited the growth of human promyelocytic leukemia HL-60 cells with an IC50 of 35 mumol/L. Incubation of HL-60 cells with 50 mumol/L trimidox for 24 hours decreased deoxyguanosine triphosphate (dGTP) and deoxycytidine triphosphate (dCTP) pools to 24% and 39% of control values, respectively. Incubation of HL-60 cells with 20 to 80 mumol/L trimidox even up to a period of 4 days did not alter the distribution of cells in different phases of cell cycle. Sequential incubation of HL-60 cells with trimidox (25 mumol/L) for 24 hours and then with 10 mumol/L tiazofurin (an inhibitor of inosine monophosphate dehydrogenase) for 4 days produced synergistic growth inhibitory activity, and the cell number decreased to 16% of untreated controls. When differentiation-linked cell surface marker expressions were determined in cells treated with trimidox and tiazofurin, a significantly increased fluorescence intensity was observed for the CD 11b (2.9-fold). CD 33 (1.9-fold), and HLA-D cell surface antigens. Expression of the transferrin receptor (CD71) increased 7.3-fold in cells treated with both agents, compared with untreated controls. Our results suggest that trimidox in combination with tiazofurin might be useful in the treatment of leukemia.

Influence of Cell Cycle Phase on Radiation-Induced Cytotoxicity and DNA Damage in Human Colon Cancer (HT29) and Chinese Hamster Ovary Cells

We have previously shown that fluorodeoxyuridine (FdUrd) radiosensitizes HT29 human colon carcinoma cells. Since treatment with FdUrd arrests cells at the G1/S-phase interface, a condition associated with increased radiation sensitivity in some cells, it seemed possible that redistribution of cells in the phases of the cell cycle might account for FdUrd-mediated radiosensitization. To begin to test this, HT29 cells were separated by centrifugal elutriation according to cell cycle phase and assessed for radiosensitivity, using a clonogenic assay, and radiation-induced DNA damage, using pulsed-field gel electrophoresis. We found that all of the elutriated fractions (which contained cells enriched in G1, G1/early S, mid to late S or G2/M phase) had the same radiation sensitivity and expressed a similar extent of radiation-induced DNA damage. To determine if the techniques used in this study could detect differences between the radiation sensitivity of cells in different phases of the cell cycle, analogous experiments were carried out using Chinese hamster ovary (CHO) cells. In contrast with the results of experiments with HT29 cells, but in agreement with previous studies, CHO cells separated under the same conditions as were used for HT29 cells showed a marked dependence on cell age of both clonogenic survival and radiation-induced DNA damage. Thus, within the limitations of the purity of separation obtained using elutriation, the radiation sensitivity of HT29 cells does not vary substantially as a function of cell cycle phase. Therefore, it seems unlikely that cell cycle redistribution alone explains the radiation sensitivity produced by exposure to FdUrd.

Induction and rejoining of radiation-induced DNA single-strand breaks: “tail moment” as a function of position in the cell cycle

Previous results using a variety of methods have shown no significant difference in induction or rejoining of radiation-induced DNA single-strand breaks (SSBs) as a function of cell-cycle position. However, concurrent of DNA damage and cell-cycle position measured using the alkaline comet assay indicates cycle-dependent differences in “tail momen” which has been shown to be a measure of SSBs. Unirradiated S phase cells showed a significantly higher tail moment presumably as a result of the presence of active replication sites. The time required to repair half of the damage appeared to be consistently shorter for G1 cells than for cells in other phases of the cell cycle (3.7 min versus approximately 5 min). Although early kinetics of rejoining (within 5 min) was identical for CHO, V79 and repair-deficient TK6 cells, TK6 cells subsequently repaired fewer breaks than did CHO or V79 cells. S phase TK6 cells rejoined SSBs more slowly than cells in the other phases; differences in rejoining rates could not be explained by the presence of a subset of slowly repairing TK6 cells. We concluide that the comet assay detects subtle differences in the tail moment when cells in different phases of the cell cycle are irradiated and allowed to repair damage. The biological importance of these observations remains to be determined.

Cytofluorometric assay for the determination of thymidine uptake and phosphorylation in living cells

Thymidine kinase is a key enzyme for the application of drugs in chemotherapy and for diagnosis. Although of great interest, its regulation during cell cycle and differentiation is difficult to study, as current techniques for isolation of cells in different phases of growth are unsatisfactory. An assay that allows the determination of enzymatic activity in situ in single cells would be much faster than present methods and would elegantly avoid synchronization procedures. We synthesized different analogues of thymidine with the 5-methyl group substituted by a fluorochrome. At least three of these compounds were phosphorylated by thymidine kinase in cell free extracts and were taken up and phosphorylated by cells in culture. The cytofluorometric signal of the accumulated fluorochrome in any given cell reflected the thymidine kinase activity of this cell. Simultaneous measurement of cell-cycle dependent parameters allowed the correlation of thymidine kinase activity with the phase of growth in mixed cell populations.

Transient c- fos expression accompanies naturally occurring cell death in the developing interhemispheric cortex of the rat

We have searched for the possible correlation of naturally occurring cell death with spontaneously enhanced c- fos expression in the developing cerebral cortex of normal Wistar albino rats. During the late prenatal and early postnatal period, cells with irregular contours and intracytoplasmic electron-dense granules (granule-containing cells) were apparent in the interhemispheric cortex, including the anterior cingulate and the retrosplenial cortices. These cells were loosely packed within the cortical layers derived from the cortical plate. Having excluded the possibility that these cells could be phagocytes by immunocytochemical experiments, we propose that they are cells in different phases of a process of autophagic degeneration and death. Images of extreme nuclear pyknosis were also apparent in identical locations. Cells showing immunoreactivity for c-Fos protein appeared in the same cortical areas. The immunoreactive cells were very abundant in the retrosplenial cortex, but were also present in the anterior cingulate cortex. These cells showed markedly irregular contours and large, densely immunoreactive intracytoplasmic inclusions; these images were similar to those of granule-containing cells revealed by conventional stains. The immunoreactivity for c-Fos protein was ephemeral, occurring exclusively during embryonic days 20 and 21, but granule-containing cells were observed for a longer period. The present results provide evidence, albeit indirect, that c- fos expression may occur in certain neural cells at the onset of a process of death by autophagia, and suggest a possible involvement of the proto-oncogene c- fos in certain forms of naturally occurring neuronal death.

Low-temperature phase transition in Cs 3D(SeO 4) 2

The lattice constants of Cs 3D(SeO 4) 2 have been measured in the temperature range 25–297 K using an automatic 4-circle X-ray diffractometer with a closed-cycle refrigerator. The changes in the lattice constants around the low-temperature phase transition ( T c3 ⋍ 180 K) seem more distinct than those in Rb 3D(SeO 4) 2, but are still very small (10 -4). The diffraction data show that the phase below T c3 is monoclinic and the space group is most probably P2 1 m . The relationship between the unit cells of different phases in Cs 3D(SeO 4) 2 and the room-temperature phase in other M 3H(XO 4) 2-type crystals is discussed.

In vitro studies of deformation and adhesion properties of transformed cells

The micropipet aspiration technique and the parallel-plate flow chamber were used to investigate the deformation and detachment properties, respectively, of normal and transformed rat fibroblasts. The normal Cloned Rat Embryo Fibroblasts (CREF) cell line was transfected with the T24 ras oncogene to produce the transformed cell line CREF T24. The CREF T24 cell line was transfected with a Kirsten ras revertant gene (K-rev 1a suppressor) to produce the CT24HKB1 cells, which have the same morphological characteristics as the cells in the CREF line. The cells utilized in this investigation were derived from the parent cell line CREF, the only differences being the presence or absence of the T24 ras oncogene and the Kirsten ras revertant gene. The detachment and deformation properties, therefore, could be related to the metastatic phenotype of the cell rather than inherent differences between disparate cell lines. Results indicated that transfecting the CREF cell line with the ras oncogene greatly modified the detachment and deformation properties. The CREF T24 cells were more easily detached from normal cells and were 50% more deformable. Both CREF and CT24HKB1 showed similar detachment properties. Based on these results, it is speculated that K-rev 1a reversed ras-induced membrane alterations in these cells. Preliminary investigations have demonstrated that both CREF and CREF T24 cells in different phases of the cell cycle differed in morphological characteristics. However, the majority of the cells within a given cell line showed similar deformation characteristics. Current investigations are focusing on characterization of both detachment and deformation properties of these cells as a function of the cell cycle using synchronization techniques.

The interaction between cells in different phases of the cell cycle and aminocarb

Synchronous cultures of Chlamydomonas segnis Ettl. were treated with aminocarb (0.5, 5.0, 10.0, or 50.0 μg ml −1) at each specific phase of the cell cycle, namely, G 1, S, G 2, and M phases. The subsequent effects of some macromolecular products were assayed at the end of the M phase. Aminocarb treatments of 0.5 μg ml −1 were at the no effects level onthe parameters monitored. However, the higher concentrations of aminocarb tested, namely, 5, 10, and 50 μg ml −1, dramatically affected not only macromolecular syntheses but also some cell cycle events. Algistatic and algicidal effects were obtained with some treatments.

Effect of Methotrexate on Cell Cycle and DNA Synthesis of Astrocytes in Primary Culture: Flow Cytometric Studies

Relatively pure population of astrocytes in primary culture were examined by flow cytometry at various time intervals after exposure to 1, 10 and 100 microM of methotrexate (MTX). The percentage population of cells in different phases of the cell cycle was determined using propidium iodide (PI) to measure cellular DNA content. DNA synthesis was assessed by measuring the fluorescence intensity of FITC-conjugated antibody to bromodeoxyuridine (BrdUrd). The two parameters were correlated to determine the location of BrdUrd-incorporating cells within the cell cycle. Exposure of astrocytes to MTX caused a consistent increase in number of cells in S-phase which correlated with the increase in BrdUrd incorporation. These studies provide supportive evidence that the astrogliosis seen in MTX encephalopathy may be due to a primary involvement of astrocytes.

Comparative study of dma content and interphase nucleolar organizer regions in human pituitary adenomas

DNA content and the number of nucleolar organizer regions (NORs) are claimed to be valuable markers of biological behavior in many tumors. Forty-three human pituitary adenomas removed by surgery were embedded in paraffin and studied by flow cytometry in order to establish DNA index and percentage of cells in different phases of cell cycle. In 31 of these tumors, silver staining showing argyrophilic proteins bound to NORs (AgNORs) was applied. DNA analysis revealed DNA aneuploidy in 32% of the adenomas. The highest number of aneuploid tumors was found in silent corticotroph adenomas (5 of 9), followed by prolactin cell adenomas (3 of 6). The lowest number of aneuploid tumors was noted in null cell adenomas and oncocytomas. The presence of an aneuploid peak could not be correlated with the size of adenomas or with invasiveness. The number of cases with S fraction higher than median was not increased in large or invasive tumors. No close correlation was found between DNA index and AgNOR reading or between S fraction and AgNOR number. These results show that DNA index and AgNOR counting must be interpreted with caution as proliferation markers in pituitary adenomas.

Functional morphology of gastrin-producing cells in different phases of periodic gastric activity in dogs

Gastrin producing cells of mucosal stomach antrum have been studied in different phases of periodical stomach activity and starvation. It has been established that functional morphology of gastrin producing cells were variable in different phases of stomach activity that coincides with the levels of gastrin in systemic blood stream.