G(1)/S but not G(0)/G(1)cell fraction is related to 5-fluorouracil cytotoxicity

BACKGROUND: Bromodeoxyuridine (BrdU) cell cycle analysis using flow cytometry is of clinical interest for making treatment decisions or for predicting response and survival, through proliferation rate (labeling index or S-phase fraction) assessment or T(pot) calculation. Thymidylate synthase expression was tested in vitro, in vivo, and clinically as a prognostic factor for 5-fluorouracil (5FU) sensitivity. However, results were still controversial. Moreover, we had reported that 5FU sensitivity was related to the labeling index of untreated cell cultures. METHODS: We used six human cancer cell lines that exhibited a wide range of 5FU sensitivity. Cell cycle analysis was performed using flow cytometry monovariate propidium iodide (PI) analysis and bivariate distributions of BrdU incorporation versus DNA content. 5FU sensitivity was assayed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) colorimetric assay. RESULTS: In all cell lines, 5FU exposure resulted in a statistically significant G(1)/S accumulation. No statistically significant relationship was seen between G(0)/G(1) delay determined by monovariate analysis and 5FU sensitivity. However, 5FU sensitivity was statistically correlated to the labeling index and G(1)/S subpopulation assessed with bivariate analysis using BrdU incorporation versus DNA content. CONCLUSIONS: Cellular proliferation parameters using BrdU incorporation are more informative than PI for in vitro 5FU sensitivity. Because BrdU incorporation could be assessed clinically, it could also be informative for 5FU clinical response prediction.     

Tylophorine arrests carcinoma cells at G1 phase by downregulating cyclin A2 expression

Tylophorine, a representative phenanthroindolizidine alkaloid from Tylophoraindica plants, exhibits anti-inflammatory and anti-cancerous growth activities. However, the underlying mechanisms of its anti-cancer activity have not been elucidated and its effects on cell cycle remain ambiguous. Here, we reveal by asynchronizing and synchronizing approaches that tylophorine not only retards the S-phase progression but also dominantly arrests the cells at G1 phase in HepG2, HONE-1, and NUGC-3 carcinoma cells. Moreover, tylophorine treatment results in down regulated cyclin A2 expression and overexpressed cyclin A2 rescues the G1 arrest by tylophorine. Thus, we are the first to report that the downregulated cyclin A2 plays a vital role in G1 arrest by tylophorine in carcinoma cells.     

c-Fos/activator protein-1 transactivates wee1 kinase at G(1)/S to inhibit premature mitosis in antigen-specific Th1 cells

M-phase promoting factor is a complex of cdc2 and cyclin B that is regulated positively by cdc25 phosphatase and negatively by wee1 kinase. We isolated the wee1 gene promoter and found that it contains one AP-1 binding motif and is directly activated by the immediate early gene product c-Fos at cellular G(1)/S phase. In antigen-specific Th1 cells stimulated by antigen, transactivation of the c-fos and wee1 kinase genes occurred sequentially at G(1)/S, and the substrate of wee1 kinase, cdc2-Tyr15, was subsequently phosphorylated at late G(1)/S. Under prolonged expression of the c-fos gene, however, the amount of wee1 kinase was increased and its target cdc2 molecule was constitutively phosphorylated on its tyrosine residue, where Th1 cells went into aberrant mitosis. Thus, an immediate early gene product, c-Fos/AP-1, directly transactivates the wee1 kinase gene at G(1)/S. The transient increase in c-fos and wee1 kinase genes is likely to be responsible for preventing premature mitosis while the cells remain in the G(1)/S phase of the cell cycle.     

Differential mRNA expression of the human DNA methyltransferases (DNMTs) 1, 3a and 3b during the G(0)/G(1) to S phase transition in normal and tumor cells

DNA methylation is essential for mammalian development, X-chromosome inactivation, and imprinting yet aberrant methylation patterns are one of the most common features of transformed cells. One of the proposed causes for these defects in the methylation machinery is overexpression of one or more of the three known catalytically active DNA methyltransferases (DNMTs) 1, 3a and 3b, yet there are clearly examples in which overexpression is minimal or non-existent but global methylation anomalies persist. An alternative mechanism which could give rise to global methylation errors is the improper expression of one or more of the DNMTs during the cell cycle. To begin to study the latter possibility we examined the expression of the mRNAs for DNMT1, 3a and 3b during the cell cycle of normal and transformed cells. We found that DNMT1 and 3b levels were significantly downregulated in G₀/G₁ while DNMT3a mRNA levels were less sensitive to cell cycle alterations and were maintained at a slightly higher level in tumor lines compared to normal cell strains. Enzymatic activity assays revealed a similar decrease in the overall methylation capacity of the cells during G₀/G₁ arrest and again revealed that a tumor cell line maintained a higher methylation capacity during arrest than a normal cell strain. These results reveal a new level of control exerted over the cellular DNA methylation machinery, the loss of which provides an alternative mechanism for the genesis of the aberrant methylation patterns observed in tumor cells.     

A Cdc28 mutant uncouples G1 cyclin phosphorylation and ubiquitination from G1 cyclin proteolysis

Proteolysis of the yeast G(1) cyclins is triggered by their Cdc28-dependent phosphorylation. Phosphorylated Cln1 and Cln2 are ubiquitinated by the SCF-Grr1 complex and then degraded by the 26 S proteasome. In this study, we identified a cak1 allele in a genetic screen for mutants that stabilize the yeast G(1) cyclins. Further characterization showed that Cln2HA was hypophosphorylated, unable to bind Cdc28, and stabilized in cak1 mutants at the restrictive temperature. Hypophosphorylation of Cln2HA could thus explain its stabilization. To test this possibility, we expressed a Cak1-independent mutant of Cdc28 (Cdc28-43244) in cak1 mutants and found that Cln2HA phosphorylation was restored, but surprisingly, the phospho-Cln2HA was stabilized. When bound to Cdc28-43244, Cln2HA was recognized and polyubiquitinated by SCF-Grr1. The Cdc28-43244 mutant thus reveals an unexpected complexity in the degradation of polyubiquitinated Cln2HA by the proteasome.     

1-Methylnicotinamide and NAD metabolism in normal and transformed normal rat kidney cells

NAD, 1-methylnicotinamide, S-adenosylmethionine, and S-adenosylhomocysteine levels were analyzed in different clones of untransformed normal rat kidney cells and in cells transformed by different viruses. No consistent changes in the levels of these metabolites were apparent as a result of malignant transformation, and also differences in the levels of metabolites did not correlate with growth rate in the various cell lines. 3-Deazaadenosine prevented synthesis of 1-methylnicotinamide but not of NAD. The S-denosylmethionine/S-adenosylhomocysteine ratio did not change in serum-starved, growth-arrested cells although 1-methylnicotinamide synthesis increased about twofold. These results were used to consider possible physiological roles for 1-methylnicotinamide. Its intracellular levels did not correlate with growth rate and were not altered by transformation. No evidence was obtained that its synthesis is involved with maintenance of nicotinamide of S-adenosylmethionine levels. Thus the biological function for 1-methylnicotinamide remains a mystery.     

Cyclic AMP delays G2 progression and prevents efficient accumulation of cyclin B1 proteins in mouse macrophage cells

In mouse macrophage cells, the increase of the intracellular cAMP level activates protein kinase A (PKA) and results in inhibition of cell cycle progression in both G1 and G2/M phases. G1 arrest is mediated by a cdk inhibitor, p27Kip1, which prevents G1 cyclin/cdk complexes from being activated in response to colony stimulating factor-1, whereas inhibition of G2/M progression has not been fully elucidated. In this report we analyzed the effect of cAMP on G2/M progression in a mouse macrophage cell line, BAC1.2F5A. Flow cytometric analysis and mitotic index measurement using both synchronized and asynchronized cells revealed that addition of cAMP-elevating agents (8-bromoadenosine 3':5'-cyclic monophosphate and 3-isobutyl-methyl-xanthine), although they did not affect S phase progression or M/G1 transition, temporarily arrested cells in G2 but eventually the cells proceeded to M phase, resulting in about 4 hours delay of G2 progression. Timing of cyclin B1/Cdc2 kinase activation was also retarded by about 4 hours, which was accompanied by inhibition of efficient accumulation of cyclin B1 proteins. Initial induction and accumulation of cyclin B1 mRNA were not hampered, but the half life of cyclin B1 proteins was significantly shorter during G2 phase in the presence of cAMP-elevating agents compared with that of the cells blocked from progressing through M phase by nocodazole. These results imply that the cAMP/PKA pathway regulates G2 phase progression by altering the stability of a crucial cell cycle regulator.     

Oxysterols induce apoptosis and accumulation of cell cycle at G(2)/M phase in the human monocytic THP-1 cell line

The cytotoxic activity of oxysterols, 7 beta-hydroxycholesterol (7 beta-OHC) and 25-hydroxycholesterol (25-OHC), has been evaluated using various leukemia cell lines. Among the tested cell lines, both oxysterols showed the highest cytotoxicity to THP-1, human monocytic leukemia cell line. These oxysterols induced apoptosis through down-regulation of Bcl-2 expression and activation of caspases. Also, the oxysterols showed the accumulation at G(2)/M phase of cell cycle through down-regulation of cyclin B1 expression. Taken together, these results indicated that both 7 beta-OHC and 25-OHC inhibited the proliferation of THP-1 cells through apoptosis and cell cycle accumulation at G(2)/M phase.     

Comparative analysis of pre-replication complex proteins in transformed and normal cells

This study examines the abundance of the major protein constituents of the pre-replication complex (pre-RC), both genome-wide and in association with specific replication origins, namely the lamin B2, c-myc, 20mer1, and 20mer2 origins. Several pre-RC protein components, namely ORC1-6, Cdc6, Cdt1, MCM4, MCM7, as well as additional replication proteins, such as Ku70/86, 14-3-3, Cdc45, and PCNA, were comparatively and quantitatively analyzed in both transformed and normal cells. The results show that these proteins are overexpressed and more abundantly bound to chromatin in the transformed compared to normal cells. Interestingly, the 20mer1, 20mer2, and c-myc origins exhibited a two- to threefold greater origin activity and a two- to threefold greater in vivo association of the pre-RC proteins with these origins in the transformed cells, whereas the origin associated with the housekeeping lamin B2 gene exhibited both similar levels of activity and in vivo association of these pre-RC proteins in both cell types. Overall, the results indicate that cellular transformation is associated with an overexpression and increased chromatin association of the pre-RC proteins. This study is significant, because it represents the most systematic comprehensive analysis done to date, using multiple replication proteins and different replication origins in both normal and transformed cell lines.     

Evidence for a direct correlation between c-Jun NH2 terminal kinase 1 activation, cyclin D2 expression, and G(1)/S phase transition in the murine hybridoma 7TD1 cells

In this study we show that the addition of fresh culture medium to high-density growth-arrested 7TD1 cells induces a strong and transient stimulation of the c-Jun NH2 terminal kinase activity (Jun kinase/JNK), a marked increase in cyclin D2 expression, the phosphorylation of pRb, and the transition from G(1) to S phase. The stimulation of cyclin D2 expression and the induction of JNK activity appear to be the consequences of the alkalinization of the extracellular medium. Indeed both parameters (i) can be induced, regardless of cell dilution, by the addition of a weak base such as triethylamine, and (ii) are together inhibited by (N-ethyl-N-isopropyl)amiloride, a specific inhibitor of the Na(+)/H(+) exchanger. We provide a strong argument indicating the existence of a direct correlation between JNK1 activation and cyclin D2 stimulation. Indeed, we demonstrate that cyclin D2 expression is blocked by SB 202190, an agent known to inhibit both JNK and p38(MAPK), but not by SB 203580, a specific inhibitor of p38(MAPK). Furthermore, we also observed that DMSO and forskolin, two agents that inhibit the proliferation of 7TD1 cells, inhibit in parallel cyclin D2 and JNK1. Altogether our results suggest that (i) JNK1 participates in the signaling pathway which controls the expression of cyclin D2 and (ii) that the inhibition of JNK1 by DMSO and forskolin could explain, at least in part, the antiproliferative action of these drugs in 7TD1 cells.     

Comparative analysis of the synchronization methods of normal and transformed human cells

Reactions of genetically identical cells to various exogenous and endogenous stimuli can vary significantly. One of the main factors of this non-genetic cellular heterogeneity is the cell cycle. The most convenient way to study the subcellular processes depending on the cell cycle stage is the synchronization of the cells. Toxic inhibitors of DNA replication and/or mitotic spindle assembly are typically used to synchronize cells. It is important to accurately select the synchronization method for a particular experiment. In this study, we performed a comparative analysis of the synchronization methods of normal and transformed human cells, paying special attention to the accuracy of synchronization and toxicity of the methods used.     

Analysis of transient G1/S arrest in E1A+E1B-19kDa transformed cells after ionizing radiation

Expression of human adenovirus type 5 E1A oncogene in normal rodent cells leads to disruption of the G1/S cell cycle arrest realization in response to DNA damage. It has been shown here that rat embryo fibroblasts transformed by E1Aad5 oncogene in complementation with E1B-19 kDa gene realize the irradiation-induced transient G1/S arrest, which depends on selective suppression of CyclinE-Cdk2 activity despite functional inactivation of p21Waf1 inhibitor. Inhibitor p21Waf1 is not revealed in complexes with cyclins E and A in E1A + E1B-19 kDa transformants, however, it is not due to p21Waf1 interaction with E1A oncoproteins, because the E1A-p21Waf1 complex formation in E1A + cHa-ras transformants does not prevent the high level of CycIE, A-p21Waf1 association. In the case of p21Waf1 inactivation, the main way of cyclin-kinase activity regulation in E1A + E1B-19 kDa cells may be Cdk2 phosphorylation. However, irradiation of E1A + E1B-19 kDa transformed cells induces no changes in CAK (Cdk7-associated) kinase activity and in the protein level of Cdc25A phosphatase, which are responsible for activating Thr160 phosphoralation and Tyr15 dephosphorylation on Cdk2. Using phospho-Tyr15-Cdk2 specific antibodies, no increase of phosphorylation at Tyr15 position on immunoprecipitated Cdk2 was detected after irradiation. It seems likely that in the case of inactivated inhibitor p21Waf1 the transient G1/S block after irradiation in E1A + E1B-19 kDa transformants depends on suppression of Cycl-E-Cdk2 activity caused by inhibition of Thr160 Cdk2 phosphorylation, but his occurs with the involvement of other kinases rather than CAK.     

Overexpression of c-Myc alters G(1)/S arrest following ionizing radiation

Study of the mechanism(s) of genomic instability induced by the c-myc proto-oncogene has the potential to shed new light on its well-known oncogenic activity. However, an underlying mechanism(s) for this phenotype is largely unknown. In the present study, we investigated the effects of c-Myc overexpression on the DNA damage-induced G(1)/S checkpoint, in order to obtain mechanistic insights into how deregulated c-Myc destabilizes the cellular genome. The DNA damage-induced checkpoints are among the primary safeguard mechanisms for genomic stability, and alterations of cell cycle checkpoints are known to be crucial for certain types of genomic instability, such as gene amplification. The effects of c-Myc overexpression were studied in human mammary epithelial cells (HMEC) as one approach to understanding the c-Myc-induced genomic instability in the context of mammary tumorigenesis. Initially, flow-cytometric analyses were used with two c-Myc-overexpressing, nontransformed immortal lines (184A1N4 and MCF10A) to determine whether c-Myc overexpression leads to alteration of cell cycle arrest following ionizing radiation (IR). Inappropriate entry into S phase was then confirmed with a bromodeoxyuridine incorporation assay measuring de novo DNA synthesis following IR. Direct involvement of c-Myc overexpression in alteration of the G(1)/S checkpoint was then confirmed by utilizing the MycER construct, a regulatable c-Myc. A transient excess of c-Myc activity, provided by the activated MycER, was similarly able to induce the inappropriate de novo DNA synthesis following IR. Significantly, the transient expression of full-length c-Myc in normal mortal HMECs also facilitated entry into S phase and the inappropriate de novo DNA synthesis following IR. Furthermore, irradiated, c-Myc-infected, normal HMECs developed a sub-G(1) population and a >4N population of cells. The c-Myc-induced alteration of the G(1)/S checkpoint was also compared to the effects of expression of MycS (N-terminally truncated c-Myc) and p53DD (a dominant negative p53) in the HMECs. We observed inappropriate hyperphosphorylation of retinoblastoma protein and then the reappearance of cyclin A, following IR, selectively in full-length c-Myc- and p53DD-overexpressing MCF10A cells. Based on these results, we propose that c-Myc attenuates a safeguard mechanism for genomic stability; this property may contribute to c-Myc-induced genomic instability and to the potent oncogenic activity of c-Myc.     

Insulin-like growth factor I triggers nuclear accumulation of cyclin D1 in MCF-7S breast cancer cells

Stimulation of the breast cancer-derived MCF-7S cell line with insulin-like growth factor I (IGF-I; 20 ng/ml) leads to enhanced expression of cyclin D1, hyperphosphorylation of pRb, DNA synthesis, and cell division. 17beta-Estradiol (E(2); 10(-9) m) is not able to stimulate proliferation of MCF-7S cells, although addition of E(2) to serum-starved cells does result in induction of cyclin D1. However, in combination with submitogenic amounts of IGF-I (2 ng/ml), E(2) induces cell proliferation. We have previously shown that the synergistic action of E(2) and IGF-I emanates from the ability of both hormones to induce cyclin D1 expression and that IGF-I action is required to induce activity of the cyclin D1-CDK4 complex, which triggers cell cycle progression. Here, we show that IGF-I (but not E(2)) is able to induce nuclear accumulation of cyclin D1 by a phosphatidylinositol 3-kinase-dependent mechanism. Nuclear accumulation of cyclin D1 and cell cycle progression were also observed when LiCl, a known inhibitor of GSK3beta, was added to E(2)-stimulated cells. Thus, inhibition of GSK3beta activity appears to trigger nuclear accumulation of cyclin D1 and cell cycle progression. This notion was confirmed by overexpression of constitutively active GSK3beta, which blocks IGF-I-induced nuclear accumulation of cyclin D1 as well as S phase transition.