Cell cycle-dependent expression of cyclooxygenase-2 in human fibroblasts.

The purpose of this investigation is to determine whether the levels of cyclooxygenase-2 (COX-2) expression are cell cycle dependent. We used a serum-starved human foreskin fibroblast model to determine changes in COX-2 mRNA, protein, and promoter activity in response to stimulation with interleukin-1b (IL-1b) and phorbol 12-myristate 13-acetate (PMA) at G0, G1, S and G2/M phases of the cell cycle. IL-1b (1 ng/ml) and PMA (100 nM) induced robust COX-2 expression in the G0 cells, and the level of COX-2 expression declined progressively after the cells had entered the cell cycle. The COX-2 mRNA level at G1, S and G2/M phases of the cell cycle was 76%, 46%, and 30% of that at G0, respectively. A 5-flanking promoter fragment of COX-2 constructed into a luciferase expression vector was transfected into cells. The promoter activity in response to PMA stimulation was significantly higher in G0 than in S phase cells. These results imply that G0 cells are the key players in inflammation and other COX-2-dependent pathophysiological processes. When the cells are in the proliferative phase, COX-2 inducibility becomes restrained probably by an endogenous control mechanism to avoid COX-2 mediated oxidative DNA damage.     

Enhanced survivability of cloned calves derived from roscovitine-treated adult somatic cells

Nuclear transfer to produce cattle is inefficient because 1) donor cells are not easily synchronized in the proper phase of the cell cycle, 2) the nucleus of these cells is not effectively reprogrammed, 3) the rate of attrition of late-term pregnancies is high, and 4) the health of early postnatal calves is compromised. The cyclin dependent kinase 2 inhibitor, roscovitine, was used to maximize cell cycle synchrony and to produce cells that responded more reliably to nuclear reprogramming. Roscovitine-treated adult bovine granulosa cells (82.4%) were more efficiently synchronized (P < 0.05) in the quiescent G0/G1 phase of the cell cycle than were serum-starved cells (76.7%). Although blastocyst development following nuclear transfer was elevated (P < 0.05) in the serum-starved group (21.1%) relative to the roscovitine-treated cells (11.8%), the number of cells in the blastocysts derived from roscovitine-treated cells was higher (P < 0.05) than those derived from the serum-starved group (roscovitine-treated group: 142.8 +/- 6.0 cells; serum-starved group: 86.8 +/- 14.5 cells). The resulting fetal and calf survival after embryo transfer was enhanced in the roscovitine-treated group (seven surviving calves from six pregnancies) compared with serum-starved controls (two calves born, one surviving beyond 60 days, from five pregnancies). Roscovitine culture can predictably synchronize the donor cell cycle and increase the nuclear reprogramming capacity of the cells, resulting in enhanced fetal and calf survival and increased cloning efficiency.     

Control of K+ influx in 3T3 cells transformed by a conditional mutant of Rous sarcoma virus

Mouse 3T3 cells transformed by a conditional mutant of Rous sarcoma virus (LA90) can assume either a normal or a transformed phenotype, depending on the temperature of cultivation. These cells (LA90) were arrested at the G0/G1 phase of the cell cycle by starvation for serum growth factors at the nonpermissive temperature (39 degrees C). Release from the G0/G1 phase by serum growth factors resulted in a rapid stimulation of Rb+ influx. To investigate whether the stimulation of Rb+ influx is obligatory for cell proliferation, the cultures were released from the G0/G1 phase by a temperature decrease in the absence of serum. A temperature decrease from 39 to 32 degrees C activated the viral pp60src gene mitogenic activity. Under these conditions, no rapid stimulation of Rb+ influx was observed. These results suggest that the rapid stimulation of Rb+ influx induced by serum growth factors is not an essential signal for cell release from the G0/G1 phase. However, a delayed increase in Rb+ influx concomitant with an increase in the cell content of K+ was observed in the cultures released from the G0/G1 phase by temperature decrease in the absence of serum growth factors. We found that the LA90 cells incubated at the permissive temperature (32 degrees C) secreted a mitogenic activity into the medium. Moreover, the conditioned medium from cultures incubated at 32 degrees C, but not at 39 degrees C, stimulate Rb+ influx in G0/G1 cells. These results indicate that Rous sarcoma virus pp60src induces a slow autocrine secretion of a mitogenic activity. This mitogenic activity slowly modulates the K+ content. Therefore, the slow elevation in cellular content of K+ is proposed to be an obligatory event for proliferation in normal and transformed cells.     

CCAAT/enhancer-binding protein delta regulates mammary epithelial cell G0 growth arrest and apoptosis.

CCAAT/enhancer-binding proteins (C/EBPs) are a highly conserved family of DNA-binding proteins that regulate cell-specific growth, differentiation, and apoptosis. Here, we show that induction of C/EBPdelta gene expression during G0 growth arrest is a general property of mammary-derived cell lines. C/EBPdelta is not induced during G0 growth arrest in 3T3 or IEC18 cells. C/EBPdelta induction is G0-specific in mouse mammary epithelial cells; C/EBPdelta gene expression is not induced by growth arrest in the G1, S, or G2 phase of the cell cycle. C/EBPdelta antisense-expressing cells (AS1 cells) maintain elevated cyclin D1 and phosphorylated retinoblastoma protein levels and exhibit delayed G0 growth arrest and apoptosis in response to serum and growth factor withdrawal. Conversely, C/EBPdelta-overexpressing cells exhibited a rapid decline in cyclin D1 and phosphorylated retinoblastoma protein levels, a rapid increase in the cyclin-dependent kinase inhibitor p27, and accelerated G0 growth arrest and apoptosis in response to serum and growth factor withdrawal. When C/EBPdelta levels were rescued in AS1 cells by transfection with a C/EBPdelta "sense" construct, normal G0 growth arrest and apoptosis were restored. These results demonstrate that C/EBPdelta plays a key role in the regulation of G0 growth arrest and apoptosis in mammary epithelial cells.     

LZ—410盼生安对肝癌细胞细胞周期时相的影响及对DNA损伤效应的观察

LZ-410盼生安是一种广谱抗肿瘤药物。本文以5-Fu作为阳性对照药物从对BEL-7402肝癌细胞周期时相的影响及对DNA损伤效应两个方面进行了初步探讨。结果表明,LZ-410能将细胞阻滞在G0/G1期,抑制了细胞的生长增殖作用。并且在本实验条件下,LZ-410并不引起DNA的损伤。这表明LZ-410抑制肝癌细胞生长增殖不是通过对DNA损伤引起的,而主要是通过阻滞肿瘤细胞于G0/G1期实现的。     

The cell cycle dependence of thermotolerance. II. CHO cells heated at 45.0 degrees C

This report extends our investigations of the cell cycle dependence of the expression of thermotolerance to include tolerance expressed by Chinese hamster ovary (CHO) cells exposed to 45.0 degrees C hyperthermia. We examined the response of asynchronous cells following exposure at 45.0 degrees C. A maximum in thermotolerance under these conditions was reached approximately 12 hr after a 15-min exposure to 45.0 degrees C hyperthermia and progressively decreased thereafter. Cells were delayed in S and G2 phase for 24 hr, after which time cell growth resumed. We then characterized the response of CHO cell populations synchronized in G1 or early or late S phase. We observed that the expression of tolerance depended on the position of cells in the cell cycle and was modulated by changes in the sensitivity of cells as they progressed through the cell cycle subsequent to the tolerance induction dose. We measured the variation in the sensitivity of these cells to 45.0 degrees C hyperthermia throughout the cell cycle and found substantial changes as cells progressed through S phase. Cells in early S phase were the most sensitive to heat at this temperature, and as these cells progressed through S phase, they became progressively more resistant. In addition, G1 cells were delayed for approximately 15 to 18 hr by a 15-min, 45.0 degrees C heat pulse, whereas S-phase cells were delayed to a lesser extent. The data presented in this report suggest that the induction of thermotolerance is relatively non-cell-cycle specific, but the magnitude of expression of tolerance depends on the position of cells in the cell cycle at the time of the subsequent challenge heat dose.     

Messenger RNAs coding for enzymes of polyamine biosynthesis are induced during the G0-G1 transition but not during traverse of the normal G1 phase

The events occurring during emergence of cells from quiescence ("G0") are not necessarily identical to those in the G1 phase of continuously dividing cells. Cellular levels of the mRNAs coding for ornithine decarboxylase (ODC) and S-adenosyl-methionine decarboxylase (SDC), key enzymes in polyamine synthesis, increased maximally within 5 h after addition of serum to resting 3T3 cells, following a kinetic course similar to that of c-myc mRNA. In a pure early G1 population of cells, prepared by centrifugal elutriation of growing fibroblasts, the levels of ODC and SDC mRNAs were not significantly lower than in other phases of the cell cycle and approximated serum-induced levels rather than the reduced values found in serum-starved cells. Thus, we conclude that the mRNAs coding for the polyamine biosynthetic enzymes, like c-myc, are growth controlled, but not regulated during traverse of a normal cell cycle.     

Analysis of cell cycle activity and population dynamics in heterogeneous plant cell suspensions using flow cytometry

Flow cytometry was used to measure cell cycle parameters in Solanum aviculare plant cell suspensions. Methods for bromodeoxyuridine (BrdU) labeling of plant nuclei were developed so that cell cycle times and the proportion of cells participating in growth could be determined as a function of culture time and conditions. The percentage of cells active in the cell cycle at 25 degrees C decreased from 52% to 19% within 7.6 d of culture; presence of a relatively large proportion of non-active cells was reflected in the results for culture growth. While the maximum specific growth rate of the suspensions at 25 degrees C was 0.34 d-1 (doubling time: 2.0 d), the specific growth rate of active cells was significantly greater at 0.67 d-1, corresponding to a cell cycle time of 1.0 d. A simple model of culture growth based on exponential and linear growth kinetics and the assumption of constant cell cycle time was found to predict with reasonable accuracy the proportion of active cells in the population as a function of time. Reducing the temperature to 17 degrees C lowered the culture growth rate but prolonged the exponential growth phase compared with 25 degrees C; the percentage of cells participating in the cell cycle was also higher. Exposure of plant cells to different agitation intensities in shake flasks had a pronounced effect on the distribution of cells within the cell cycle. The proportion of cells in S phase was 1.8 times higher at a shaker speed of 160 rpm than at 100 rpm, while the frequency of G0 + G1 cells decreased by up to 27%. Because of the significant levels of intraculture heterogeneity in suspended plant cell systems, flow cytometry is of particular value in characterizing culture properties and behavior.     

Effects of foscarnet on the cell kinetics of Madin-Darby canine kidney cells

The antiherpes compound, foscarnet (trisodium phosphonoformate), showed concentration-dependent effects on the cell kinetics of Madin-Darby canine kidney cells. At 1 mM, only minor effects could be seen on cell proliferation and cell cycle distribution, as measured by flow cytometry DNA analysis. Treatment with 5 mM foscarnet resulted in an accumulation of cells in the S-phase although no complete cell cycle block was evident. At 10 mM foscarnet, cells accumulated earlier in the S phase, probably at the G1/S border. However, at both 5 and 10 mM foscarnet the block was not established until after 15 h incubation. Upon removing 10 mM foscarnet after 24 h incubation, G1 cells rapidly entered the S phase, whereas the progression through S and G2 + M was delayed considerably. The DNA synthesizing S phase seems, therefore, to be the main cell cycle phase affected by foscarnet.     

Growth Conditions and Cell Cycle Phase Modulate Phase Transition Temperatures in RBL-2H3 Derived Plasma Membrane Vesicles

Giant plasma membrane vesicle (GPMV) isolated from a flask of RBL-2H3 cells appear uniform at physiological temperatures and contain coexisting liquid-ordered and liquid-disordered phases at low temperatures. While a single GPMV transitions between these two states at a well-defined temperature, there is significant vesicle-to-vesicle heterogeneity in a single preparation of cells, and average transition temperatures can vary significantly between preparations. In this study, we explore how GPMV transition temperatures depend on growth conditions, and find that average transition temperatures are negatively correlated with average cell density over 15°C in transition temperature and nearly three orders of magnitude in average surface density. In addition, average transition temperatures are reduced by close to 10°C when GPMVs are isolated from cells starved of serum overnight, and elevated transition temperatures are restored when serum-starved cells are incubated in serum-containing media for 12h. We also investigated variation in transition temperature of GPMVs isolated from cells synchronized at the G1/S border through a double Thymidine block and find that average transition temperatures are systematically higher in GPMVs produced from G1 or M phase cells than in GPMVs prepared from S or G1 phase cells. Reduced miscibility transition temperatures are also observed in GPMVs prepared from cells treated with TRAIL to induce apoptosis or sphingomyelinase, and in some cases a gel phase is observed at temperatures above the miscibility transition in these vesicles. We conclude that at least some variability in GPMV transition temperature arises from variation in the local density of cells and asynchrony of the cell cycle. It is hypothesized that GPMV transition temperatures are a proxy for the magnitude of lipid-mediated membrane heterogeneity in intact cell plasma membranes at growth temperatures. If so, these results suggest that cells tune their plasma membrane composition in order to control the magnitude of membrane heterogeneity in response to different growth conditions.     

The cell cycle dependence of thermotolerance. I. CHO cells heated at 42 degrees C

We examined the dependence of heat killing and thermotolerance on the position and progression of Chinese hamster ovary (CHO) cells in the cell cycle. We measured cell cycle perturbations and survival of asynchronous and synchronized G1-, S-, and G2-phase cells resulting from continuous heating at 42.0 degrees C for up to 80 hr. Thermotolerance under these conditions was transient in nature, was dependent on the position of cells in the cell cycle, and occurred concurrently with a heat-induced delay of progression of G1- and G2-phase cells. When G1 cells were heated, survival decreased to 25% after 4 hr, at which time the thermotolerance was expressed. For G2 cells survival decreased initially at the same rate (T0 congruent to 3 hr) but thermotolerance was not expressed until approximately 12 hr, at which time the survival was 4%. The rate of decrease in survival was much more rapid for cells heated in mid-S phase (T0 congruent to 0.5 hr), and these cells did not express thermotolerance at a measurable level. Concurrent with the expression of thermotolerance, the progression of cells heated in G1 and G2 was delayed. Following the expression of tolerance, progression resumed at a rate approximately equal to the rate of decrease in survival of the G1 population. Cells heated in mid-S phase continued to progress through the cell cycle until they reached G2, where they were also delayed.     

Cell cycle analysis of cultured porcine mammary cells

One of the major points of debate in determining the effectiveness of nuclear transfer technology has been the phase of the cell cycle of the donor cell at the time of nuclear transfer. Here, a primary mammary cell line has been isolated and various treatments for synchronization of the cell cycle have been tested. The cells were then simultaneously stained for DNA content and protein content and the percentages of cells in G1, G0, S, and G2 + M were estimated. In the first experiment, cells were either cycling, grown to confluence, or serum-starved for 5 days. Serum starvation increased (p < 0.05) the percentage of cells in G0 compared to confluent or cycling cells from 3% to 8% to 22%. By using forward scatter to determine the size of the cells it was determined that if small cells (7-15 microm) were selected from the serum-starved group 43.9% will be in G(0) as compared to 4.5% of cycling cells and 9.9% of confluent cells. Dimethyl sulfoxide (DMSO) treatment (0%, 0.5%, or 1.0%) for 72 hours (shown to synchronize some cell types in G0) had no effect on the percentage of cells in G0, G1, S, or G2 + M. Treatment with mimosine (0 microM, 0.4 microM, 0.8 microM or 1.2 microM), a compound that should synchronize the cells in G1, increased (p < 0.05) the percentage of cells in G1 from 66.7% (0 microM mimosine) to 79.0% to 82.0%. Finally, treatment with colchicine for 24 hours (shown to synchronize some cell types in G2 + M) increased (p < 0.05) the percentage of cells in G2 + M (0 microM colchicine) from 13.3% to 27.2% to 31.6%. It is concluded that many cell cycle synchronization techniques are effective in porcine mammary cell lines, but none of the techniques are 100% effective. Such results should help elucidate the mechanisms involved in nuclear transfer.     

cAMP-mediated growth inhibition of a B-lymphoid precursor cell line Reh is associated with an early transient delay in G2/M, followed by an accumulation of cells in G1

This study was undertaken to gain more insight into the effects of cyclic adenosine monophosphate (cAMP) on cell-cycle progression in the B-lymphoid precursor cell line Reh. The adenylate cyclase activator forskolin reduced the proliferation of asynchronously growing Reh cells by 50% after 72 hr culture. Growth inhibition was associated with an accumulation of cells in G1. Furthermore, we demonstrated that forskolin provoked a delay of cells for approximately 10 hr in G2/M prior to the G1 arrest. Two different methods were applied to elucidate how cells in different phases of the cell cycle were affected by an elevated cAMP level. One method was based on centrifugal elutriation, whereby synchronous cell populations from the different phases of the cell cycle were isolated. By the other method, S-phase cells were selectively stained by pulsing asynchronously growing cells with bromo-deoxyuridine (BrdU). The data demonstrate that the position of a cell in the cell cycle is critical in determining how the cell will respond to an elevated cAMP level. Thus cells in G1 at the time forskolin is added are not delayed in G2/M, but they will subsequently accumulate in G1 after 48 hr. Cells given forskolin in G2/m, however, are delayed for 10 hr in G2/M, but they do not accumulate in G1. Cells given forskolin in the S phase are delayed in G2/M as well as arrested in G1. The results suggest that cAMP inhibits growth of the Reh cells by preventing the cells from passing important restriction points located in the G1 and G2 phases of the cell cycle.     

Downregulation of JNK/SAPK activity is associated with the cross-resistance to P-glycoprotein-unrelated drugs in multidrug-resistant FM3A/M cells overexpressing P-glycoprotein

In the present study, cross-drug resistance in multidrug-resistant (MDR) cells, which overexpress P-glycoprotein (Pgp), a mdr1 gene product, against Pgp-unrelated drugs, and its relevance to c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) activity were examined. The multidrug-resistant FM3A/M cells overexpressing Pgp were resistant to apoptotic cell death induced either by Pgp-related drugs including vincristine and vinblastine, which are pumped out by Pgp, or by the Pgp-unrelated drugs including 5'-fluorouracil (5-FU) and bleomycin, which are not targets for Pgp, compared with the parental FM3A cells. Verapamil reversed the resistance of FM3A/M cells to apoptosis induced by the Pgp-related drugs but not that induced by the Pgp-unrelated drugs. Interestingly, FM3A/M cells have shown significantly lower basal and drug-stimulated JNK/SAPK activities than FM3A cells. After transfection with pEBG-SEK or pEBG-SAPK constructs, FM3A/M cells recovered the basal and Pgp-unrelated drug-stimulated activities of JNK/SAPK and the susceptibility to Pgp-unrelated drug-induced apoptotic cell death comparable to those of FM3A cells. Furthermore, FM3A cells became resistant to apoptotic cell death induced by vincristine and 5-FU after transfection with pEBG-SEK(K --> R), a dominant negative inhibitory mutant of SEK. These results suggest that downregulation of JNK/SAPK activity appears to confer on Pgp-associated FM3A/M cells a cross-resistance to Pgp-unrelated drugs.     

Flow cytometric cell cycle analysis of cultured brown bear fibroblast cells

The aim of this study was to assess by flow cytometry the cell cycle of brown bear fibroblast cells cultured under different growth conditions. Skin biopsies were taken in Cantabria (Spain) from a live, anaesthetized brown bear. DNA analysis was performed by flow cytometry following cell DNA staining with propidium iodide. Serum starvation increased (P<0.01) the percentage of G0/G1 phase cells (92.7+/-0.86) as compared to cycling cells (39.7+/-0.86) or cells cultured to confluency (87.3+/-0.86). DMSO included for 48h in the culture significantly increased (P<0.01) the percentage of G0/G1 phase of the cell cycle at all concentrations used and decreased percentages of S phase in a dose-dependent fashion. Roscovitine increased the G0/G1 phase of the cell cycle (P<0.01) at 15microM concentration. Interestingly, the G2/M stage significantly increased at 30 and 50microM compared to the control and 15microM (P<0.02). The cell cycle of brown bear adult fibroblast cells can be successfully synchronized under a variety of culture conditions.     

Speedy/Ringo C regulates S and G2 phase progression in human cells

Cyclin-dependent kinases (CDKs) control cell cycle transitions and progression. In addition to their activation via binding to cyclins, CDKs can be activated via binding to an unrelated class of cell cycle regulators termed Speedy/Ringo (S/R) proteins. Although mammals contain at least five distinct Speedy/Ringo homologues, the specific functions of members of this growing family of CDK activators remain largely unknown. We investigated the cell cycle roles of human Speedy/Ringo C in HEK293 cells. Down-regulation of Speedy/Ringo C by RNA interference delayed S and G2 progression whereas ectopic expression had the opposite effect, reducing S and G2/M populations. Double thymidine arrest and release experiments showed that overexpression of Speedy/Ringo C promoted late S phase progression. Using a novel three-color FACS protocol to determine the length of G2 phase, we found that the suppression of Speedy/Ringo C by RNAi prolonged G2 phase by ~30 min whereas ectopic expression of Speedy/Ringo C shortened G2 phase by ~25 min. In addition, overexpression of Speedy/Ringo C disrupted the G2 DNA damage checkpoint, increased cell death and caused a cell cycle delay at the G1-to-S transition. These observations indicate that CDK-Speedy/Ringo C complexes positively regulate cell cycle progression during the late S and G2 phases of the cell cycle.     

Cell kinetic status of mouse lens epithelial cells lacking αA- and αB-crystallin

alphaA- and alphaB-crystallins are small heat shock proteins and molecular chaperones that are known to prevent non-specific aggregation of denaturing proteins. Recent work indicates that alphaA-/- lens epithelial cells grow at a slower rate than wild-type cells, and cultured alphaB-/- cells demonstrate increased hyperproliferation and genomic instability, suggesting that these proteins may exert a direct effect on the cell cycle kinetics, and influence cell proliferation. However, the cell cycle parameters of alphaA/alphaBKO (double knockout) cells have not been analyzed. Here we investigate the cell cycle kinetics of synchronized mouse lens epithelial cultures derived from wild-type and alphaA/alphaB double knockout (alphaA/alphaBKO) mice using BrdU labeling of proliferating cells, and flow cytometric analysis. We also provide data on the changing pattern of expression of HSP25, a small heat shock protein in alphaA/alphaBKO and wild-type cells during the cell cycle. Using serum starvation to synchronize cells in the quiescent G0 phase, and restimulation with serum followed by BrdU labeling and flow cytometry, the data indicated that as compared to wild-type cells, a <50% smaller fraction of the alphaA/alphaBKO cells entered the DNA synthetic S phase of the cell cycle. Furthermore, there was a delay in cell cycle transit through S phase in alphaA/alphaBKO cells, suggesting that although capable of entering S phase, the alphaA/alphaBKO cells are blocked in G1 phase, and are delayed in their cell cycle progression. Immunoblot analysis with antibodies to the small heat shock protein HSP25 indicated that although HSP25 increased in G1 phase of wild-type cells, and remained elevated on further progression through the cell cycle, HSP25 accumulation was delayed to S phase in alphaA/alphaBKO cells. These data can be interpreted to indicate that mouse lens epithelial cell progression through the cell cycle is significantly affected by expression of alphaA and alphaB-crystallin.     

Inhibition of growth of ASL-1w murine leukemia cells by anti-thymus leukemia antigen (TL) serum in the absence of complement.

Previous studies have shown that the cell surface expression of thymus leukemia antigen (TL) on ASL-1w leukemia cells varies with the progression of the cells through the growth cycle. Expression of TL is maximal in S phase, and its quantitative expression varies directly with DNA synthesis. In the present study, the effect of anti-TL serum on the growth of ASL-1w cells was examined. The antiserum, tested in the absence of complement, affected the growth of these cells in biphasic manner. When the antiserum concentration was 0.1% or greater, there was a rapid decline in the rate of DNA synthesis, and after 5 to 7 hr, cell death. When the antiserum concentration was 0.067% or less, the decline in the rate of synthesis of DNA did not become apparent until 5 to 6 hr after treatment. Under these conditions, there was approximately a 20% increase in cell number after 24 hr of culture. The hypothesis that treatment of ASL-1w cells with the lesser concentration of anti-TL serum blocks the cells in G2 phase of the cell cycle is discussed.     

Sensitization to the cytotoxicity of adriamycin by verapamil and heat in multidrug-resistant Chinese hamster ovary cells.

Development of multidrug resistance to anticancer agents is a major limitation for the success of cancer chemotherapy. The chemosensitizer verapamil increases intracellular accumulation of drugs such as adriamycin in certain multidrug-resistant cell lines. When combined with verapamil, hyperthermia should be able to alter membrane permeability to adriamycin and to enhance the cytotoxicity of the drug. Verapamil increased the cytotoxicity of adriamycin in multidrug-resistant Chinese hamster ovary cells (CH(R)C5) but not in drug-sensitive cells (AuxB1). Hyperthermia (42 degrees C) alone clearly increased the cytotoxicity of adriamycin in AuxB1 cells. There was also a small increase in CH(R)C5 cells at 42 and 43 degrees C. In drug-resistant cells, the cytotoxicity of adriamycin increased considerably when verapamil was combined with heat. This effect was dependent on temperature and increased with time of incubation. At 37 degrees C, verapamil increased the uptake of adriamycin in CH(R)C5 cells, while drug efflux decreased. When verapamil was combined with hyperthermia, drug efflux decreased even further. These results led to an overall increase in intracellular accumulation of the drug. In drug-sensitive cells, hyperthermia increased both the uptake and efflux of adriamycin, but verapamil had no effect. Verapamil plus heat increased the cytotoxicity of adriamycin in drug-resistant cells, and this was accompanied by altered permeability of the membrane to the drug. Hyperthermia combined with verapamil could be beneficial by increasing the effectiveness of adriamycin in the elimination of multidrug-resistant cells in a localized target region.     

DNA damage during G2 phase does not affect cell cycle progression of the green alga Scenedesmus quadricauda

DNA damage is a threat to genomic integrity in all living organisms. Plants and green algae are particularly susceptible to DNA damage especially that caused by UV light, due to their light dependency for photosynthesis. For survival of a plant, and other eukaryotic cells, it is essential for an organism to continuously check the integrity of its genetic material and, when damaged, to repair it immediately. Cells therefore utilize a DNA damage response pathway that is responsible for sensing, reacting to and repairing damaged DNA. We have studied the effect of 5-fluorodeoxyuridine, zeocin, caffeine and combinations of these on the cell cycle of the green alga Scenedesmus quadricauda. The cells delayed S phase and underwent a permanent G2 phase block if DNA metabolism was affected prior to S phase; the G2 phase block imposed by zeocin was partially abolished by caffeine. No cell cycle block was observed if the treatment with zeocin occurred in G2 phase and the cells divided normally. CDKA and CDKB kinases regulate mitosis in S. quadricauda; their kinase activities were inhibited by Wee1. CDKA, CDKB protein levels were stabilized in the presence of zeocin. In contrast, the protein level of Wee1 was unaffected by DNA perturbing treatments. Wee1 therefore does not appear to be involved in the DNA damage response in S. quadricauda. Our results imply a specific reaction to DNA damage in S. quadricauda, with no cell cycle arrest, after experiencing DNA damage during G2 phase.