Sensitivity to macrophage-mediated cytostasis is cell cycle dependent

We have examined the sensitivity of proliferating lymphoid cells in different phases of the cell cycle to macrophage-mediated cytostatic activity. These studies evaluated the ability of target cells enriched in individual cell cycle phases to pass into the next phase during brief (2–6 hr) periods of coculture with activated or nonactivated peritoneal macrophages. Both normal (concanavalin A-stimulated spleen cells) and neoplastic (Gross virus-induced thymic lymphoma) cells were analyzed. Spleen cells or lymphoma cells were first separated by centrifugal elutriation into populations highly enriched for G₁, S, or G₂/M phases of the cell cycle and cultured in the presence of nonactivated or activated macrophages for periods of 2, 4, or 6 hr. The cellular DNA content of recovered nonadherent target cells was then analyzed by flow cytometry after staining with propidium iodide. Macrophage contamination of target cell populations was insignificant under these conditions. Nonactivated macrophages did not affect target cell cycle traverse when compared with target cells cultured alone. Activated macrophage mediated cytostatic activity resulted in complete block of the transition of cells in G₁ phase into S phase and of the further accumulation of DNA by cells in early S phase. Cells already in mid to late S phase were able to continue DNA replication at rates nearly equivalent to control cells. There was no inhibition of the passage of cells through G₂ or mitosis. These effects were seen by as early as 2 hr of macrophage-target cell coculture and both normal and neoplastic cells exhibited identical patterns of cell cycle phase sensitivity.     

Interferon affects cell growth progression by modulating DNA polymerases activity

A multiparametric analysis of the effects of human recombinant interferon alpha type A on Daudi cells involving flow cytometry and in vitro analysis of alpha and beta DNA polymerase activities has been performed. Results have disclosed (within 60 min of interferon treatment) a decrease of alpha polymerase driven DNA synthesis persisting to at least 24 h, while beta polymerase was poorly affected. Moreover, after 24 h of interferon treatment, a reduction of BrdUrd incorporation per cell, assessed by flow cytometry, was observed suggesting that DNA synthesis in S phase cells is almost completely abolished. The analysis of the effect of interferon on the distribution of cell cycle phases indicated that the G1/S transition is not inhibited by the treatment. These results support the hypothesis that interferon generates a transient initiating signal which quickly reaches the nucleus and produces a rapid inhibition of alpha polymerase activity, leading finally to the slowing of cell cycle progression.     

Studies of Schizosaccharomyces pombe DNA polymerase alpha at different stages of the cell cycle.

The status of Schizosaccharomyces pombe (fission yeast) DNA polymerase alpha was investigated at different stages of the cell cycle. S.pombe DNA polymerase alpha is a phosphoprotein, with serine being the exclusive phosphoamino acid. By in vivo pulse labeling experiments DNA polymerase alpha was found to be phosphorylated to a 3-fold higher level in late S phase cells compared with cells in the G2 and M phases, but the steady-state level of phosphorylation did not vary significantly during the cell cycle. Tryptic phosphopeptide mapping demonstrated that the phosphorylation sites of DNA polymerase alpha from late S phase cells were not the same as that from G2/M phase cells. DNA polymerase alpha partially purified from G1/S cells had a different mobility in native gels from that from G2/M phase cells. The partially purified polymerase alpha from G1/S phase cells had a higher affinity for single-stranded DNA than that from G2/M phase cells. Despite the apparent differences in cell cycle-dependent phosphorylation, mobility in native gels and affinity for DNA, the in vitro enzymatic activity of the partially purified DNA polymerase alpha did not appear to vary during the cell cycle. The possible biological significance of these cell cycle-dependent characteristics of DNA polymerase alpha is discussed.     

Mimosine reversibly arrests cell cycle progression at the G1-S phase border

It has previously been demonstrated that the compound mimosine inhibits cell cycle traverse in late G1 phase prior to the onset of DNA synthesis (Hoffman BD, Hanauske-Abel HM, Flint A, Lalande M: Cytometry 12:26-32, 1991; Lalande M: Exp Cell Res 186:332-339, 1990). These results were obtained by using flow cytometric analysis of DNA content to compare the effects of mimosine on cell cycle traverse with those of aphidicolin, an inhibitor of DNA polymerase alpha activity. We have now measured the incorporation of bromodeoxyuridine into lymphoblastoid cells by flow cytometry to determine precisely where the two inhibitors act relative to the initiation of DNA synthesis. It is demonstrated here that mimosine arrests cell cycle progression at the G1-S phase border. The onset of DNA replication occurs within 15 min of releasing the cells from the mimosine block. In contrast, treatment with aphidicolin results in the accumulation of cells in early S phase. These results indicate that mimosine is a suitable compound for affecting the synchronous release of cells from G1 into S phase and for analyzing the biochemical events associated with this cell cycle phase transition.     

The effects of dehydroaltenusin, a novel mammalian DNA polymerase alpha inhibitor, on cell proliferation and cell cycle progression

As described previously, a natural product isolated from fungus (Acremonium sp.), dehydroaltenusin, is an inhibitor of mammalian DNA polymerase alpha in vitro [Y. Mizushina, S. Kamisuki, T. Mizuno, M. Takemura, H. Asahara, S. Linn, T. Yamaguchi, A. Matsukage, F. Hanaoka, S. Yoshida, M. Saneyoshi, F. Sugawara, K. Sakaguchi, Dehydroaltenusin, a mammalian DNA polymerase alpha inhibitor, J. Biol. Chem. 275 (2000) 33957_33961]. In this study, we investigated the interaction of dehydroaltenusin with lipid bilayers using an in vitro liposome system, which is a model of the cell membrane, and found that approximately 4% of dehydroaltenusin was incorporated into liposomes. We also investigated the influence of dehydroaltenusin on cultured cancer cells. Dehydroaltenusin inhibited the growth of HeLa cells with an LD50 value of 38 microM, and as expected, S phase accumulation in the cell cycle. The total DNA polymerase activity of the extract of incubated cells with dehydroaltenusin was 23% lower than that of nontreated cells. Dehydroaltenusin increased cyclin E and cyclin A levels. In the analysis of the cell cycle using G1/S synchronized cells by employing hydroxyurea, the compound delayed both entry into the S phase and S phase progression. In a similar analysis using G2/M synchronized cells by employing nocodazole, the compound accumulated the cells at G1/S and inhibited entry into the S phase. Thus, the pharmacological abrogation of cell proliferation by dehydroaltenusin may prove to be an effective chemotherapeutic agent against tumors.     

Productive HIV-1 infection of macrophages restricted to the cell fraction with proliferative capacity.

Retroviruses establish productive infection only in proliferating cells. Macrophages are often considered to be non-proliferating in vitro yet are susceptible to HIV-1 infection. This has led to the conclusion that HIV-1 can establish infection independent of host cell proliferation. We here report that a small proportion of macrophages does have proliferative capacity. A comparable small fraction of monocyte derived macrophages (MDM) supported productive HIV-1 infection as demonstrated in limiting dilution culture. Fluorescence activated cell sorting on the basis of incorporation of BrdUrd, a thymidine analog, and subsequent PCR analysis revealed the presence of proviral DNA only in the BrdUrd positive cell fraction with DNA synthesizing activity. To identify which phase of cell cycle is required for establishment of productive infection, growth arrest in G1 or G1/S phase prior to inoculation was performed. gamma-Irradiation, which arrests primary cells in G1, prevented both cell proliferation and establishment of productive infection in MDM. Treatment of MDM with aphidicolin, a specific inhibitor of DNA polymerase alpha and delta which arrests cells in G1/S phase of the cell cycle, also inhibited DNA synthesis but did not prevent establishment of productive infection which is completely analogous to observations in T cells. Our data thus indicate that not cell division itself but cellular conditions that coincide with cell proliferation are apparently indispensable for establishment of productive infection.     

The Stress-activated Protein Kinase Hog1 Mediates S Phase Delay in Response to Osmostress

Control of cell cycle progression by stress-activated protein kinases (SAPKs) is essential for cell adaptation to extracellular stimuli. Exposure of yeast to osmostress activates the Hog1 SAPK, which modulates cell cycle progression at G1 and G2 by the phosphorylation of elements of the cell cycle machinery, such as Sic1 and Hsl1, and by down-regulation of G1 and G2 cyclins. Here, we show that upon stress, Hog1 also modulates S phase progression. The control of S phase is independent of the S phase DNA damage checkpoint and of the previously characterized Hog1 cell cycle targets Sic1 and Hsl1. Hog1 uses at least two distinct mechanisms in its control over S phase progression. At early S phase, the SAPK prevents firing of replication origins by delaying the accumulation of the S phase cyclins Clb5 and Clb6. In addition, Hog1 prevents S phase progression when activated later in S phase or cells containing a genetic bypass for cyclin-dependent kinase activity. Hog1 interacts with components of the replication complex and delays phosphorylation of the Dpb2 subunit of the DNA polymerase. The two mechanisms of Hog1 action lead to delayed firing of origins and prolonged replication, respectively. The Hog1-dependent delay of replication could be important to allow Hog1 to induce gene expression before replication.     

Cytostatic activity of in vitro generated macrophages: evidence for a prostaglandin-independent reversible cytostatic mechanism

Culture of spleen cells for 5 days has previously been shown to result in the generation of strongly adherent cells from nonadherent precursors. In the current report it is shown that the majority (85-95%) of adherent cells are Mac-1+, FcR+, Thy 1.2- macrophages. Expression of effector activity by these macrophages requires exposure to activating signals. Coculture of the macrophages with Con A-stimulated spleen cells results in the expression of cytostatic activity against lymphocytic and monocytic tumor cell lines. Significant cytostatic activity is apparent within 6 hr after addition of the activating cells. Culture supernates of Con A-stimulated spleen cells (CAS-CM) are not effective in inducing cytostatic activity in the adherent macrophage population either alone or in the presence of additional Con A. However, stimulation of the culture generated macrophages with LPS in the presence of CAS-CM does induce cytostatic activity. The effector cell must be metabolically active in order to effect cytostasis insofar as heat fixation of the culture generated macrophage population eliminates effector activity. Proliferation of the tumor cells is significantly reduced after a 4-hr incubation period with the activated macrophages and is reduced two- to threefold after an 8- to 12-hr incubation period. The cytostatic effect is rapidly reversible. Proliferative activity of the tumor cells returned to control level within 12-24 hr after removal from activated macrophages. Cell cycle analysis indicated that the target cells were not arrested in a single stage of cell cycle, although an increase in frequency of cells in G1-phase was observed. Fluorescence analysis of bromodeoxyuridine (BrdU) incorporation rate demonstrated that the rate of DNA synthesis was reduced in all of the cells in the target population and that the mean rate of BrdU incorporation of the inhibited cells was three- to fivefold lower than control cells. RNA and protein synthesis were not affected to the same degree as DNA synthesis. The cytostatic effect was not mediated by prostaglandins or thymidine insofar as addition of indomethacin and 2-deoxycytidine did not prevent the cytostatic activity of the macrophages. The supernates of activated macrophages contained little inhibitory activity especially when indomethacin was included in the culture medium (19% inhibition of tumor cell proliferation by 1:1 dilution of supernate). The activity that was present could be eliminated by dialysis against fresh culture medium using Spectropor membranes with a 1000-Da molecular cutoff.(ABSTRACT TRUNCATED AT 400 WORDS)     

The cell cycle-coupled expression of topoisomerase IIalpha during S phase is regulated by mRNA stability and is disrupted by heat shock or ionizing radiation.

Topoisomerase II is a multifunctional protein required during DNA replication, chromosome disjunction at mitosis, and other DNA-related activities by virtue of its ability to alter DNA supercoiling. The enzyme is encoded by two similar but nonidentical genes: the topoisomerase IIalpha and IIbeta genes. In HeLa cells synchronized by mitotic shake-off, topoisomeraseII alpha mRNA levels were found to vary as a function of cell cycle position, being 15-fold higher in late S phase (14 to 18 h postmitosis) than during G1 phase. Also detected was a corresponding increase in topoisomerase IIalpha protein synthesis at 14 to 18 h postmitosis which resulted in significantly higher accumulation of the protein during S and G2 phases. Topoisomerase IIalpha expression was not dependent on DNA synthesis during S phase, which could be inhibited without effect on the timing or level of mRNA expression. Mechanistically, topoisomerase IIalpha expression appears to be coupled to cell cycle position mainly through associated changes in mRNA stability. When cells are in S phase and mRNA levels are maximal, the half-life of topoisomerase IIalpha mRNA was determined to be approximately 30 min. A similar decrease in mRNA stability was also induced by two external factors known to delay cell cycle progression. Treatment of S-phase cells, at the time of maximum topoisomerase IIalpha mRNA stability, with either ionizing radiation (5 Gy) or heat shock (45 degrees C for 15 min) caused the accumulated topoisomerase IIalpha mRNA to decay. This finding suggests a potential relationship between stress-induced decreases in topoisomerase IIalpha expression and cell cycle progression delays in late S/G2.     

Phosphoinositide 3-kinase signaling overrides a G2 phase arrest checkpoint and promotes aberrant cell cycling and death of hematopoietic cells after DNA damage

DNA damage activates arrest checkpoints to halt cell cycle progression in G1 and G2 phases. These checkpoints can be overridden in hematopoietic cells by cytokines, such as erythropoietin, through the activation of a phosphoinositide 3-kinase (PI3K) signaling pathway. Here, we show that PI3K activity specifically overrides delayed mechanisms effecting permanent G1 and G2 phase arrests, but does not affect transient checkpoints arresting cells up to 10 hours after gamma-irradiation. Assessing the status of cell cycle regulators in hematopoietic cells arrested after gamma-irradiation, we show that Cdk2 activity is completely inhibited in both G1 and G2 arrested cells. Despite the absence of Cdk2 activity, cells arrested in G2 phase did retain detectable levels of Cdk1 activity in the absence of PI3K signaling. However, reactivation of PI3K promoted robust increases in both Cdk1 and Cdk2 activity in G2-arrested cells. Reactivation of Cdks was accompanied by a resumption of cell cycling, but with strikingly different effectiveness in G1 and G2 phase arrested cells. Specifically, G1-arrested cells resumed normal cell cycle progression with little loss in viability when PI3K was activated after gamma-irradiation. Conversely, PI3K activation in G2-arrested cells promoted endoreduplication and death of the entire population. These observations show that cytokine-induced PI3K signaling pathways promote Cdk activation and override permanent cell cycle arrest checkpoints in hematopoietic cells. While this activity can rescue irradiated cells from permanent G1 phase arrest, it results in aberrant cell cycling and death when activated in hematopoietic cells arrested at the G2 phase DNA damage checkpoint.     

S-Phase-specific activation of PKC alpha induces senescence in non-small cell lung cancer cells

Protein kinase C (PKC) has been widely implicated in positive and negative control of cell proliferation. We have recently shown that treatment of non-small cell lung cancer (NSCLC) cells with phorbol 12-myristate 13-acetate (PMA) during G1 phase inhibits the progression into S phase, an effect mediated by PKC delta-induced up-regulation of the cell cycle inhibitor p21 Cip1. However, PMA treatment in asynchronously growing NSCLC cells leads to accumulation of cells in G2/M. Studies in post-G1 phases revealed that PMA induced an irreversible G2/M cell cycle arrest in NSCLC cells and conferred morphological and biochemical features of senescence, including elevated SA-beta-Gal activity and reduced telomerase activity. Remarkably, this effect was phase-specific, as it occurred only when PKC was activated in S, but not in G1, phase. Mechanistic analysis revealed a crucial role for the classical PKC alpha isozyme as mediator of the G2/M arrest and senescence, as well as for inducing p21(Cip1) an obligatory event for conferring the senescence phenotype. In addition to the unappreciated role of PKC isozymes, and specifically PKC alpha, in senescence, our data introduce the paradigm that discrete PKCs trigger distinctive responses when activated in different phases of the cell cycle via a common mechanism that involves p21 Cip1 up-regulation.     

The effect of in vitro heat exposure on the recovery of nuclear matrix-bound DNA polymerase alpha activity during the different phases of the cell cycle in synchronized HeLa S3 cells.

HeLa S3 cells were synchronized by a double thymidine block or aphidicolin treatment and the levels of nuclear matrix-bound DNA polymerase alpha activity were then measured using activated calf thymus DNA as template. The nuclear matrix was obtained by 2 M NaCl extraction and DNase I digestion of isolated nuclei incubated at 37 degrees C for 45 min prior to subfractionation. In all phases of the cell cycle 25-30% of nuclear DNA polymerase alpha activity remained matrix-bound, even when cells were in the G1 phase. No dynamic association of DNA polymerase alpha activity with the matrix was seen, at variance with previous results obtained in regenerating rat liver. The variations measured in matrix-bound activity closely followed those detected in isolated nuclei throughout the cell cycle. If nuclei were not heat-stabilized very low levels of DNA polymerase alpha activity were measured in the matrix (1-2% of total nuclear activity). Heat incubation of nuclei failed to produce any enrichment in matrix-associated newly replicated DNA, whereas the sulfhydryl cross-linking chemical sodium tetrathionate did. Therefore the results obtained after the heat stabilization procedure do not completely fit with the model that envisions the nuclear matrix as the active site where eucaryotic DNA replication takes place.     

Inhibition of endothelial cell proliferation by platelet factor-4 involves a unique action on S phase progression

Modulation of endothelial cell proliferation and cell cycle progression by the "chemokine" platelet factor-4 (PF-4) was investigated. PF-4 inhibited DNA synthesis, as well as proliferation of endothelial cells derived from large and small blood vessels. Inhibition by PF-4 was independent of the type and the concentration of stimuli used for the induction of endothelial cell proliferation. Inhibition of cell growth by PF-4 was reversible. The effects of PF-4 were antagonized by heparin. Cell cycle analysis using [3H]thymidine pulse labeling during traverse of synchronous cells from G0/G1 to S phase revealed that addition of PF-4 during G1 phase completely abolished the entry of cells into S phase. In addition, PF-4 also inhibited DNA synthesis in cells that were already in S phase. In exponentially growing cells, addition of PF-4 resulted in an accumulation of > 70% of the cells in early S phase, as determined by FACS (Becton-Dickinson Immunocytometry Systems, Mountain View, CA). In cells synchronized in S phase by hydroxyurea and then released, addition of PF-4 promptly blocked further progression of DNA synthesis. These results demonstrate that in G0/G1-arrested cells, PF-4 inhibited entry of endothelial cells into S phase. More strikingly, our studies have revealed a unique mode of endothelial cell growth inhibition whereby PF-4 effectively blocked cell cycle progression during S phase.     

Nuclear metabolic changes induced by tumor necrosis factor in Daudi lymphoma cells; a multiparametric analysis.

The effects of r-TNF alpha on cell cycle progression and DNA polymerase activity in Daudi lymphoma cells have been analyzed. Cytofluorimetric analysis of the cell cycle after 6 to 24 hr of treatment revealed both a decrease of BrdU incorporation per cell and a light inhibition of S phase as assessed by the analysis of the percentual distribution of cell cycle compartments. The reduction of BrdU incorporation can be related to the early decrease in the rate of DNA synthesis that follows r-TNF alpha treatment. These results suggest that one of the early events induced by r-TNF alpha at nuclear level is the slowering of DNA synthesis leading to a reduced cell cycle progression.     

Further characterization of a murine temperature-sensitive mutant, tsFT20 strain, containing heat-labile DNA polymerase alpha-activity

tsFT20 cells, which have temperature-sensitive DNA polymerase alpha-activity, were characterized mainly at the cellular level. The cells lost their ability to synthesize DNA immediately after a shift to non-permissive temperature. The extent of decrease in the activity of DNA polymerase alpha in whole-cell extracts was the same as that of the decrease in the DNA replication ability determined by [3H]thymidine incorporation. At 39 degrees C, tsFT20 cells lost most of their colony-forming ability in one doubling time (16 h). The cells could not grow at higher than 38 degrees C, but could grow at 37 degrees C. When tsFT20 cells were synchronized at the G1/S boundary and incubated at 39 degrees C, they could not complete the S phase, ceasing cell cycle progression in mid-S phase. A temperature shift (33 degrees C----39 degrees C) experiment indicated that the whole S phase was temperature-sensitive, whereas the G2 and M phases were not. These results confirmed that DNA polymerase alpha plays a key role in DNA replication in mammalian cells.     

用双参数流式细胞光度术(FCM)研究阿克拉霉素对L_(1210)白血病细胞周期的影响

本文用双参数FCM技术,对同一个细胞的DNA和RNA含量进行相关测量,比较了ACM B对小鼠L_(1210)白血病细胞周期和RNA含量的影响.结果发现在一次给药后8小时可导致早、中期S的积累,并抑制S期细胞的DNA合成;到24小时DNA合成恢复正常,并进入G_2期,但由于G_2期细胞进入M期受阻,造成G_2期细胞的积累,这时被阻断在G_2期的细胞RNA含量显著增加,形成正不平衡生长,而给药剂量较大的实验组(1/1.5LD_(50))S期细胞的RNA含量不随着DNA含量的增加而增加,形成负不平衡生长,ACM A和ACM B对体内Li_(210)细胞周期作用相同.     

Mode of estrogen action on cell proliferation in CAMA-1 cells: II. Sensitivity of G1 phase population

The mammary cancer cell line CAMA-1 synchronized at the G1/S boundary by thymidine block or at the G1/M boundary by nocodazole was used to evaluate 1) the sensitivity of a specific cell cycle phase or phases to 17 beta-estradiol (E2), 2) the effect of E2 on cell cycle kinetics, and 3) the resultant E2 effect on cell proliferation. In synchronized G1/S cells, E2-induced 3H-thymidine uptake, which indicated a newly formed S population, was observed only when E2 was added during, but not after, thymidine synchronization. Synchronized G2/M cells, enriched by Percoll gradient centrifugation to approximately 90% mitotic cells, responded to E2 added immediately following selection; the total E2-treated population traversed the cycle faster and reached S phase approximately 4 hr earlier than cells not exposed to E2. When E2 was added during the last hour of synchronization (ie, at late G2 or G2/M), or for 1 hr during mitotic cell enrichment, a mixed response occurred: a small portion had an accelerated G1 exit, while the majority of cells behaved the same as controls not incubated with E2. When E2 addition was delayed until 2 hr, 7 hr, or 12 hr following cell selection, to allow many early G1 phase cells to miss E2 exposure, the response to E2 was again mixed. When E2 was added during the 16 hr of nocodazole synchronization, when cells were largely at S or possibly at early G2, it inhibited entry into S phase. The E2-induced increase or decrease of S phase cells in the nocodazole experiments also showed corresponding changes in mitotic index and cell number. These results showed that the early G1 phase and possibly the G2/M phase are sensitive to E2 stimulation, late G1, G1/S, or G2 are refractory; the E2 stimualtion of cell proliferation is due primarily to an increased proportion of G1 cells that traverse the cell cycle and a shortened G1 period, E2 does not facilitate faster cell division; and estrogen-induced cell proliferation or G1/S transition occurs only when very early G1 phase cells are exposed to estrogen. These results are consistent with the constant transition probability hypothesis, that is, E2 alters the probability of cells entering into DNA synthesis without significantly affecting the duration of other cell cycle phases. Results from this study provide new information for further studies aimed at elucidating E2-modulated G1 events related to tumor growth.     

Calmodulin regulates DNA polymerase alpha activity during proliferative activation of NRK cells.

When Normal Rat Kidney cells are allowed to reenter the cell cycle after quiescence they start to replicate DNA around 12 h, reaching a maximum at 20 h. Activation of DNA polymerase alpha parallels the increase in DNA synthesis. The addition of two different anti-calmodulin drugs, trifluoroperazine (7.5 microM) or W13 (10 micrograms/ml), to the media at 4 h after proliferative activation, inhibits DNA synthesis by 55% and 80%, respectively. The blockade of calmodulin produced by trifluoroperazine allows the cells to progress through G1 phase but stops progression through S phase as determined by 5-Bromo deoxyuridine labeling. Both anti-calmodulin drugs also inhibit by more than 50% the increase in DNA polymerase alpha activity observed at 20 h. These results indicate that a calmodulin-dependent event, essential for the activation of DNA polymerase alpha and subsequently for DNA replication, is produced during G1. Therefore, the control of DNA polymerase alpha activation is one of the ways by which calmodulin is regulating the progression of NRK cells through S phase.