Number Of Cells In S-phase Research Articles

Thrombin Enhances Spontaneous Tumor Growth and Cell Cycle Activation by Downregulation of p27Kip1 and Upregulation of Skp2 and MiR-222

Although it has been generally accepted that hypercoagulability contributes to enhancing tumor growth via generation of thrombin (Cancer Cell 10:355, 2006), it has not been rigorously proven, nor has the mechanism been established at the cell cycle level. Previous studies have employed thrombin-treated tumor cell lines in vitro and in vivo. In vitro studies were performed in the presence of serum which contains a panoply of growth factors. In vivo studies have used huge non-pathologic concentrations of tumor cells injected into the flank, organ or blood of a mouse. In these situations, tumor growth could be a result of thrombin-induced angiogenesis. We therefore employed a transgenic mouse prostate cancer model (TRAMP) programmed to develop prostate CA over a period of 140–175 days. We treated these animals with thrombin to induce hypercoagulability or hirudin to inhibit endogenous thrombin production, to determine whether thrombin regulates this process independent of angiogenesis. Repetitive thrombin injection enhanced prostate tumor volume 6–8 fold (p<0.04). Repetitive hirudin decreased tumor volume 13–24 fold (p<0.04) via its effect on generated endogenous thrombin, n=6. Thrombin enhanced the production of several vascular growth factors and receptors 2.5 – 3 fold in the liver (VEGF, KDR, ANG-2, Tie2, GRO-1, CD31) and enhanced angiogenesis in the liver, n=3–4. Thrombin had no effect on tumor angiogenesis. Thus, the thrombin-induced spontaneous tumor growth was independent of angiogenesis. We next turned our attention to cell cycle regulators in serum-starved (72 hr) Go-synchronized LNcap prostate CA cells, employing Brdu and Propidium iodide staining. Addition of thrombin (0.5 u/ml) or its PAR-1 receptor agonist, TFLLRN (100 uM) had the same effect as androgen containing serum, inducing cells to leave Go, enter G1 and progress to S-phase. At 8 hrs the number of S-phase cells increased dramatically for both the serum (29 fold) as well as thrombin-treated cells (48 fold), n=3. Similar observations were noted in a Glioblastoma cell line, T98G. We further analyzed the effect of thrombin by performing immunoblots on cell cycle components mediated during cell growth and proliferation. In synchronized Go cells, levels of p27Kip1, a cyclin-dependent kinase inhibitor are high, while levels of cyclins D1 and A, the activation subunits for cyclin-dependent kinases are low. Both thrombin or serum addition led to down-regulation of p27Kip1 with concomitant induction of Skp2, the E3 ubiquitin ligase for p27Kip1. Cyclins D1 and A are induced by similar kinetics, indicating entry into S-phase by 8 hrs. Since p27Kip1 appears to be a rate-limiting down-regulator of the cell cycle (absent with high tumor grade and predicts poor prognosis), we confirmed its role by testing the effect of thrombin or TFLLRN by transfecting p27Kip1 in LNcap cells. This transfection completely prevented the cell cycle stimulation induced by these agonists. A similar approach was used with Skp2 knock down (KD), a negative down-regulator of p27Kip1. KD of Skp2 (over expressed in numerous cancers) completely prevented cell cycle progression induced by thrombin/TFLLRN. MiRNA 222 (upregulated in many cancers) is another down-regulator of p27Kip1. Further analysis following thrombin treatment revealed a robust upregulation at 4 and 8 hrs, providing further proof for the role of thrombin in down-regulating p27Kip1 and stimulating tumor cell entrance into S-phase. Thus, 1) Thrombin enhances spontaneous prostate cell growth in vivo in the absence of enhanced angiogenesis; 2) Thrombin activates the tumor cell cycle by stimulating the down-regulation of p27Kip1 through the upregulation of Skp2 and MiRNA 222.

SALL4 is a key regulator of survival and apoptosis in human leukemic cells

AbstractIncreasing studies suggest that SALL4 may play vital roles in leukemogenesis and stem cell phenotypes. We have mapped the global gene targets of SALL4 using chromatin immunoprecipitation followed by microarray hybridization and identified more than 2000 high-confidence, SALL4-binding genes in the human acute promyelocytic leukemic cell line, NB4. Analysis of SALL4-binding sites reveals that genes involved in cell death, cancer, DNA replication/repair, and cell cycle were highly enriched (P < .05). These genes include 38 important apoptosis-inducing genes (TNF, TP53, PTEN, CARD9, CARD11, CYCS, LTA) and apoptosis-inhibiting genes (Bmi-1, BCL2, XIAP, DAD1, TEGT). Real-time polymerase chain reaction has shown that expression levels of these genes changed significantly after SALL4 knockdown, which ubiquitously led to cell apoptosis. Flow cytometry revealed that reduction of SALL4 expression in NB4 and other leukemia cell lines dramatically increased caspase-3, annexin V, and DNA fragmentation activity. Bromodeoxyuridine-incorporation assays showed decreased numbers of S-phase cells and increased numbers of G1- and G2-phase cells indicating reduced DNA synthesis, consistent with results from cell proliferation assays. In addition, NB4 cells that express low levels of SALL4 have significantly decreased tumorigenecity in immunodeficient mice. Our studies provide a foundation in the development of leukemia stem cell–specific therapy by targeting SALL4.

MiR-221 controls CDKN1C/p57 and CDKN1B/p27 expression in human hepatocellular carcinoma

The identification of target mRNAs is a key step for assessing the role of aberrantly expressed microRNAs in human cancer. MiR-221 is upregulated in human hepatocellular carcinoma (HCC) as well as in other malignancies. One proven target of miR-221 is CDKN1B/p27, whose downregulation affects HCC prognosis. Here, we proved that the cyclin-dependent kinase inhibitor (CDKI) CDKN1C/p57 is also a direct target of miR-221. Indeed, downregulation of both CDKN1B/p27 and CDKN1C/p57 occurs in response to miR-221 transfection into HCC-derived cells and a significant upregulation of both CDKN1B/p27 and CDKN1C/p57 occurs in response to antimiR-221 transfection. A direct interaction of miR-221 with a target site on the 3' UTR of CDKN1C/p57 mRNA was also demonstrated. By controlling these two CDKIs, upregulation of miR-221 can promote growth of HCC cells by increasing the number of cells in S-phase. To assess the relevance of these studies in primary tumors, matched HCC and cirrhosis samples were assayed for miR-221, for CDKN1B/p27 and CDKN1C/p57 expression. MiR-221 was upregulated in 71% of HCCs, whereas CDKN1B/p27 and CDKN1C/p57 proteins were downregulated in 77% of cases. A significant inverse correlation between miR-221 and both CDKN1B/p27 and CDKN1C/p57 was found in HCCs. In conclusion, we suggest that miR-221 has an oncogenic function in hepatocarcinogenesis by targeting CDKN1B/p27 and CDKN1C/p57, hence promoting proliferation by controlling cell-cycle inhibitors. These findings establish a basis toward the development of therapeutic strategies aimed at blocking miR-221 in HCC.

Effect of a small molecule inhibitor of the JAK2/STAT3 pathway on self-renewal of glioblastoma stem cells

2003 Background: Recent data indicate that initiation and maintenance of several cancers including glioblastoma (GBM) may be driven by a small subset of cells called tumor stem cells. A key feature necessary to maintain stem cells is self-renewal. However, the mechanisms underlying GBM stem cell (GSC) self-renewal remain unknown. We identified STAT3 as a key transcription factor associated with poor outcome and treatment resistance in GBM. STAT3 is also an important regulator of self-renewal in several stem cell types. We thus tested the hypothesis that targeting STAT3 with a novel small molecule inhibitor would block GSC self-renewal. Methods: GSC neurosphere cultures from 12 human GBM tumors were expanded under serum free conditions. Self-renewal of GSCs was quantified by neurosphere formation efficiency. Synthesis and analysis of over 150 caffeic acid analogs was performed to identify the novel and potent STAT3 inhibitor WP1193. Results: Western blot analysis demonstrated that STAT3 was activated and phosphorylated (ser727 and tyr705) at high levels in 10 out of 12 GSC neurosphere cultures undergoing self-renewal. Treatment of GSCs with WP1193 at increasing concentrations from 0 to 10uM resulted in a dose dependent decrease in cell proliferation and neurosphere formation in all GSC lines tested, with an associated decrease in both total and phosphorylated STAT3. Treatment with WP1193 at 5uM for 48 hrs was sufficient to completely eliminate self-renewal and neurosphere formation at 14 days under limiting dilution conditions versus untreated controls (control neurosphere formation efficiency 12–40%). Quantitative RT-PCR and FACS analysis of CD133, a putative marker of GBM stem cells, showed a significant depletion of CD133 positive cells after WP1193 treatment (>5uM). Cell cycle analysis demonstrated that WP1193 treatment also significantly reduced the number of cells in S-Phase and significantly increased G1 arrest compared to matched controls of untreated or temozolomide treated GSCs. Conclusions: These data indicate that STAT3 is potentially important regulator of GSC self-renewal that is also associated treatment resistance in GBM. WP1193 is a potent inhibitor of GSC self-renewal and is a promising agent for therapeutically targeting GSCs. No significant financial relationships to disclose.

A study of the localization and accumulation of S-phase cells in the retina of newt Pleurodeles waltl after experimental pigment epithelial detachment

This work continues the studies of the proliferative ability of cells in the adult newt retina. The model of experimental detachment of the retina from pigment epithelium and two techniques to saturate the ocular tissues in vivo with precursors of DNA synthesis were used: (1) the method of repeated [3H]-thymidine labeling and subsequent autoradiographic analysis of semithin sections and (2) an original method for continuous labeling of thymidine analog bromodeoxyuridine and subsequent immunochemical detection. The data obtained confirm and extend our previous data on the localization of DNA-synthesizing cells in the neural retina and expose the pattern of S-phase cell accumulation after retinal detachment for each proliferation-competent cell population. In addition to cells in the growth zone of the retina, Muller glia, microglia, and minor cell population in the vitreal part of interneurons, DNA-synthesizing cells included astrocytes of the optic nerve and cells of its vascular network. Four weeks after detachment, the number of S-phase cells in the growth zone could reach 15-20%, while the above-mentioned DNA-synthesizing cells in the differentiated retina have low reproductive rate and could produce only one generation within the same period.

A cyclic chimeric interferon-α2b peptide induces apoptosis in tumor cells

Interferons alpha (IFNsα) are a family of related proteins exhibiting antiviral, antiproliferative and immunoregulatory activities. Although IFNsα have been widely employed for the pharmacological treatment of different types of cancer, the therapeutic efficacy occasionally can be diminished by the appearance of side effects, neutralizing antibodies or tumor resistance. In the search of mimetic peptides of the IFN-α2b molecule, we have recently synthesized a chimeric cyclic peptide that inhibits IFN-α2b binding to its receptor and exerts an IFN-like antiproliferative activity. In order to study the mechanism of growth inhibition of the cyclic chimera, we evaluated its ability to induce cell cycle arrest or apoptosis in WISH cells. We found that the chimeric peptide did not cause a cell cycle arrest, although the entire IFN-α2b molecule did modify cell cycle by increasing the number of S-phase cells. In spite of this difference, both molecules were able to induce apoptosis through the activation of caspases 8 and 9, indicating the involvement of death receptor and mitochondrial pathways. In addition, both peptidic derivative and IFN-α2b altered the expression of Bcl-2 family proteins and induced the release of cytochrome C to cytosol, supporting the participation of mitochondrial pathway in the induction of apoptosis. In conclusion, we demonstrated that the chimeric cyclic peptide behaved as a potent inducer of apoptosis and it could be a potentially useful agent for the treatment of certain malignancies.

Induction of Microphthalmia Transcription Factor (Mitf) by Forskolin and Stimulation of Melanin Release in UISO-Mel-6 Cells

Microphthalmia transcription factor (Mitf) is implicated as the master gene for survival of melanocytes, as well as a key transcription factor regulating the expression of major melanogenic proteins. We have shown that Mitf is a novel prognostic marker in patients with melanoma and that it plays a role in melanoma differentiation. Melanocyte-stimulating hormone up-regulates Mitf expression. Forskolin is a known alpha-melanocyte-stimulating hormone agonist. This study was undertaken to determine if forskolin induces differentiation in UISO-Mel-6 cell lines. Forskolin inhibits cell proliferation and increases the expression of Mitf and tyrosinase related protein-1. It also increases extracellular melanin production. In addition, we showed by flow cytometry that forskolin decrease the number of cells in S-phase without engaging the cells in apoptosis. Our results suggest that Forskolin induces differentiation in melanoma cells via Mitf pathway.

Mitochondria as determinant of nucleotide pools and chromosomal stability

Mitochondrial function plays an important role in multiple human diseases and mutations in the mitochondrial genome have been detected in nearly every type of cancer investigated to date. However, the mechanism underlying the interrelation is unknown. We used human cell lines depleted of mitochondrial DNA as models and analyzed the outcome of mitochondrial dysfunction on major cellular repair activities. We show that the deoxyribonucleoside triphosphate (dNTP) pools are affected, most prominently we detect a 3-fold reduction of the dTTP pool when normalized to the number of cells in S-phase. It is known that imbalanced dNTP pools are mutagenic and in accordance, we show that mitochondrial dysfunction results in chromosomal instability, which can explain its role in tumor development. We did not find any straightforward correlation between ATP levels and dNTP pools in cells with defective mitochondrial activity. Our results suggest that mitochondria are central players in maintaining genomic stability and in controlling essential nuclear processes such as upholding a balanced supply of nucleotides.

Early proliferation of umbilical cord blood cells from premature neonates

Human umbilical cord blood (UCB) is an important source of haematopoietic stem cells; however, the behaviour of progenitor cells obtained from premature and full-term neonates is still a controversy subject. Thus, the aim of this study was to evaluate cell cycle parameters and the proliferative capacity of UCB progenitor cells from premature and full-term neonates. Clonogenic assays were performed with methylcellulose, medium supplemented with recombinant stimulating growth factors and the colonies were scored on the seventh day and the 14th day of culture. A cell cycle study was carried out by DNA analysis using flow cytometry and 30 000 events were acquired; p107 and p130 expressions were analysed by Western blotting. Cultures obtained from UCB of premature neonates showed an early growth of colony-forming unit (CFU)-burst forming unit erythroid/CFU-granulocyte, erythrocyte, macrophage and megakaryocyte (BFU-E/GEMM), and CFU-granulocyte, macrophage (GM) by the seventh day of culture (P < 0.001). Therefore, the number and morphological characteristics of these colonies were comparable with those obtained from full-term neonates, on the 14th day of culture. At the 14th day, a large amount of CFU-GM was detected in the premature group (P < 0.0032). The premature culture on the 14th day showed fibroblasts and was comparable to those of full-term neonates on the 21st day in terms of number and morphology of the colonies. DNA analysis showed that the number of cells in S-phase was also higher in premature samples when compared to full-term neonates, P < 0.0021 (0 h = 12.8 vs. 2.5%; 16 h = 10.5 vs. 5.9%; 20 h = 13.5 vs. 10.3%; 24 h = 13.8 vs. 9.1%; 48 h = 14.0 vs. 5.4%; 72 h = 20.5 vs. 8.9%; 96 h = 13.8 vs. 7.7%). The Western blotting results demonstrated that p107 and p130 cell cycle protein expressions were higher in premature cells than in full-term cells. These results suggest that the higher capacity of proliferation and early differentiation of premature UCB might not be related only to the amount of stem/progenitor cells but also to a different timing of cell cycle entry.

The environmental carcinogen 3-nitrobenzanthrone and its main metabolite 3-aminobenzanthrone enhance formation of reactive oxygen intermediates in human A549 lung epithelial cells

The environmental contaminant 3-nitrobenzanthrone (3-NBA) is highly mutagenic and a suspected human carcinogen. We aimed to evaluate whether 3-NBA is able to deregulate critical steps in cell cycle control and apoptosis in human lung epithelial A549 cells. Increased intracellular Ca 2+ and caspase activities were detected upon 3-NBA exposure. As shown by cell cycle analysis, an increased number of S-phase cells was observed after 24 h of treatment with 3-NBA. Furthermore, 3-NBA was shown to inhibit cell proliferation when added to subconfluent cell cultures. The main metabolite of 3-NBA, 3-ABA, induced statistically significant increases in tail moment as judged by alkaline comet assay. The potential of 3-NBA and 3-ABA to enhance the production of reactive oxygen species (ROS) was demonstrated by flow cytometry using 2′,7′-dichlorofluorescein-diacetate (DCFH-DA). The enzyme inhibitors allopurinol, dicumarol, resveratrol and SKF525A were used to assess the impact of metabolic conversion on 3-NBA-mediated ROS production. Resveratrol decreased dichlorofluorescein (DCF) fluorescence by 50%, suggesting a role for CYP1A1 in 3-NBA-mediated ROS production. Mitochondrial ROS production was significantly attenuated (20% reduction) by addition of rotenone (complex I inhibition) and thenoyltrifluoroacetone (TTFA, complex II inhibition). Taken together, the results of the present study provide evidence for a genotoxic potential of 3-ABA in human epithelial lung cells. Moreover, both compounds lead to increased intracellular ROS and create an environment favorable to DNA damage and the promotion of cancer.

Cyclin D1 Overexpression Increases Chemosensitivity Via the Induction of Bim in Myeloma Cells.

Recent study analyzing the gene expression in myeloma cells using cDNA microarray showed that myeloma cells are divided into 7 groups. Two showed a high expression of cyclin D1 together with a low expression of cyclin D2, and they belong to the low-risk group when analyzed based on event-free and overall survival. Since cyclin D1 promotes the cell cycle progression, we sought to explain why cyclin D1 overexpression appears to be a favorable prognostic variable for multiple myeloma (MM) patients treated with high-dose chemotherapy and single or double autologous transplantation. It is not clear whether the down-regulation of cyclin D2 in these myeloma cells might offset cyclin D1 overexpression in cell biology. We have established a myeloma cell line (RPMI8226) with cyclin D1 overexpression by transfection of the cyclin D1 gene using a retrovirus vector to analyze biological changes in myeloma cells with cyclin D1 overexpression. In comparison with myeloma cells transfected with the vector only (mock), the analysis of gene expression by cDNA microarray showed the down-regulation of cyclin D2 and MCL-1 and the up-regulation of CED6 in cyclin D1-transfected myeloma cells. However, there were no significant changes in Bcl-related genes between them. And as we expected, cyclin D1-transfected myeloma cells showed high proliferative activity, increased number of cells in S-phase, and increased pRb protein. Next we analyzed their sensitivity for bortezomib (Millennium Pharmaceuticals, Inc., USA), immunomodulatory thalidomide analogs (IMiD1, D2, D3) (Celgene Corporation, USA) and dexamethasone. Bortezomib and dexamethasone induced apoptosis at an earlier time point (12hr) in cyclin D1 transfectant compared to mock transfectant, concomitant with decreased expression of MCL-1, but with increased expression of Bim. Furthermore, we confirmed in cell culture condition with a low concentration of FCS that these results were not due to just promotion of the cell cycle caused by cyclin D1 overexpression. Given that myeloma cells with cyclin D1 overexpression easily undergo apoptosis upon bortezomib or dexamethasone treatment, this may explain why patients with those myeloma cells are in the low-risk group. With this finding, we then analyzed the expression of cyclin D1 by RQ-PCR and immunocytostaining before and after 2 or 3 cycles of VAD (vincristine, doxorubicin, dexamethasone). There was no significant difference between the response to VAD and the reduction rate of cyclin D1 positive myeloma cells or the decrease of cyclin D1 expression in RQ-PCR. Interestingly, cyclin D2 expression increases relatively in progressive disease (PD) after chemotherapy containing high-dose melphalan followed by autologous stem cell transplantation compared with those of cyclin D1 and D3 by RQ-PCR. Therefore, cyclin D1-induced chemosensitivity may be due to the induction of Bim, which consequently inhibits the function of MCL-1. New strategies to down-regulate of MCL-1 might be useful in the treatment of MM.

Regeneration in Macrostomum lignano (Platyhelminthes): cellular dynamics in the neoblast stem cell system

Neoblasts are potentially totipotent stem cells and the only proliferating cells in adult Platyhelminthes. We have examined the cellular dynamics of neoblasts during the posterior regeneration of Macrostomum lignano. Double-labeling of neoblasts with bromodeoxyuridine and the anti-phospho histone H3 mitosis marker has revealed a complex cellular response in the first 48 h after amputation; this response is different from that known to occur during regeneration in triclad platyhelminths and in starvation/feeding experiments in M. lignano. Mitotic activity is reduced during the first 8 h of regeneration but, at 48 h after amputation, reaches almost twice the value of control animals. The total number of S-phase cells significantly increases after 1 day of regeneration. A subpopulation of fast-cycling neoblasts surprisingly shows the same dynamics during regeneration as those in control animals. Wound healing and regeneration are accompanied by the formation of a distinct blastema. These results present new insights, at the cellular level, into the early regeneration of rhabditophoran Platyhelminthes.

Examination of granule layer cell count, cell density, and single‐pulse brdu incorporation in rat organotypic hippocampal slice cultures with respect to culture medium, septotemporal position, and time in vitro

Adult neurogenesis in the dentate gyrus is assuming an increasingly important role in supporting hippocampal-dependent learning and the modulation of mood and anxiety. Moreover, injury to the developing postnatal dentate gyrus has profound effects on neurogenesis and hippocampal learning throughout life. Organotypic hippocampal slice cultures represent an attractive model for studying neurogenesis both in the early postnatal and adult hippocampus, as they retain much of their anatomical and functional circuitry in vitro. Ongoing neurogenesis has been recently demonstrated in organotypic hippocampal slice cultures. However, cell proliferation, one of the critical components of neurogenesis, has yet to be characterized in this culture system. We examined single-pulse S-phase bromo-deoxyuridine (BrdU) labeling in the dentate granule layer with respect to the septotemporal position of origin of the slice culture, the medium the cultures were grown in, and the time the cultures were maintained in vitro up to 14 days, when they are believed to have matured to a near adult state. Using single 10-microm sections through a culture as our reference volume, we report significant effects of septotemporal position on the number of granule layer cells and the number of cells in S-phase, as estimated by short-survival (2 hours) BrdU studies. We report a declining rate of BrdU incorporation, evidence of significant structural changes within the granule cell layer, and differences in cell death between culture media over the first 14 days in vitro. We report caution with the use of BrdU cell density and changes in cell number to indirectly estimate proliferation.

Identification and characterization of NYGGF4, a novel gene containing a phosphotyrosine-binding (PTB) domain that stimulates 3T3-L1 preadipocytes proliferation

A novel gene named NYGGF4, which was expressed at a higher level in obese subjects, was isolated and characterized. It is a 1527-bp cDNA, containing 753 nucleotides of an ORF (open reading frame) predicting 250 amino acids with a molecular mass of 28.27 kDa. Amino acid sequence analysis revealed NYGGF4 has a phosphotyrosine-binding (PTB) domain. Northern blot analysis revealed NYGGF4 is expressed primarily in adipose tissue, heart, and skeletal muscle but not in any other tissue examined. Confocal imagery analyses with green fluorescent protein-tagged protein transiently expressed in 3T3-L1 preadipocytes and 293-T cells show that NYGGF4 localizes in the cytoplasm. Furthermore, ectopic expression of NYGGF4 dramatically increases the proliferation of 3T3-L1 peadipocytes without affecting adipocytic differentiation. And the NYGGF4 expression 3T3-L1 cells had a greater number of cells in S-phase. Our data suggest that NYGGF4 might be a signaling molecule and could play a role in cell growth and adipogenesis process.

Short fluorodeoxyuridine exposure of different human glioblastoma lines induces high-level accumulation of S-phase cells that avidly incorporate 125I-iododeoxyuridine

Radio-iododeoxyuridine (IdUrd) is a potential Auger radiation therapy agent incorporated into DNA during the synthesis phase. In this study we sought to optimise S-phase targeting by modulating cellular cycling and radio-IdUrd DNA incorporation using short non-toxic fluorodeoxyuridine (FdUrd) incubations. Three human glioblastoma cell lines with different p53 expression were pre-treated with various FdUrd conditions. After different intervals, (125)I-IdUrd DNA incorporation was measured. Fluorescence-activated cell sorter cell cycle analysis was performed after identical intervals post FdUrd pre-treatment. The highest increase in (125)I-IdUrd DNA incorporation was induced by 1-h incubation with 1 muM FdUrd. Increase in radio-IdUrd DNA incorporation was greatest 16-24 h after FdUrd, reaching factors of >or=7.5 over baseline incorporation in the three cell lines. Furthermore, cell synchronisation in S phase was observed with a peak of >or=69.5% in the three cell lines at 16 and 24 h post FdUrd, corresponding to an increase of 2.5-4.1 over baseline. FdUrd-induced thymidine synthesis inhibition led to S-phase accumulation that was maximal after an interval of 16-24 h and time-correlated with the highest radio-IdUrd DNA incorporation. These observations might allow the rational design of an Auger radiation therapy targeting a maximal number of S-phase cells in single treatment cycles.

The Schizosaccharomyces pombe Replication Inhibitor Spd1 Regulates Ribonucleotide Reductase Activity and dNTPs by Binding to the Large Cdc22 Subunit

Ribonucleotide reductase (RNR) is an essential enzyme that provides the cell with a balanced supply of deoxyribonucleoside triphosphates for DNA replication and repair. Mutations that affect the regulation of RNR in yeast and mammalian cells can lead to genetic abnormalities and cell death. We have expressed and purified the components of the RNR system in fission yeast, the large subunit Cdc22p, the small subunit Suc22p, and the replication inhibitor Spd1p. It was proposed (Liu, C., Powell, K. A., Mundt, K., Wu, L., Carr, A. M., and Caspari, T. (2003) Genes Dev. 17, 1130-1140) that Spd1 is an RNR inhibitor, acting by anchoring the Suc22p inside the nucleus during G1 phase. Using in vitro assays with highly purified proteins we have demonstrated that Spd1 indeed is a very efficient inhibitor of fission yeast RNR, but acting on Cdc22p. Furthermore, biosensor technique showed that Spd1p binds to the Cdc22p with a KD of 2.4 microM, whereas the affinity to Suc22p is negligible. Therefore, Spd1p inhibits fission yeast RNR activity by interacting with the Cdc22p. Similar to the situation in budding yeast, logarithmically growing fission yeast increases the dNTP pools 2-fold after 3 h of incubation in the UV mimetic 4-nitroquinoline-N-oxide. This increase is smaller than the increase observed in budding yeast but of the same order as the dNTP pool increase when synchronous Schizosaccharomyces pombe cdc10 cells are going from G1 to S-phase.

Diabetic mice are protected from normally lethal nephrotoxicity of S-1,2-dichlorovinyl-l-cysteine (DCVC): role of nephrogenic tissue repair

Streptozotocin (STZ)-induced diabetic (DB) rats are protected from nephrotoxicity of gentamicin, cisplatin and mercuric chloride, although the mechanisms remain unclear. Ninety percent of DB mice receiving a LD90 dose (75 mg/kg, ip) of S-1,2-dichlorovinyl-l-cysteine (DCVC) survived in contrast to only 10% of the nondiabetic (NDB) mice surviving the same dose. We tested the hypothesis that the mechanism of protection is upregulated tissue repair. In the NDB mice, DCVC produced steep temporal increases in blood urea nitrogen (BUN) and plasma creatinine, which were associated with proximal tubular cell (PTC) necrosis, acute renal failure (ARF), and death within 48 h. In contrast, in the DB mice, BUN and creatinine increased less steeply, declining after 36 h to completely resolve by 96 h. HPLC analysis of plasma and urine revealed that DB did not alter the toxicokinetics of DCVC. Furthermore, activity of renal cysteine conjugate beta-lyase, the enzyme that bio-activates DCVC, was unaltered in DB mice, undermining the possibility of lower bioactivation of DCVC leading to lower injury. [3H]-thymidine pulse labeling and PCNA analysis indicated an early onset and sustained nephrogenic tissue repair in DCVC-treated DB mice. BRDU immunohistochemistry revealed a fourfold increase in the number of cells in S-phase in the DB kidneys even without exposure to DCVC. Blocking the entry of cells into S-phase by antimitotic intervention using colchicine abolished stimulated nephrogenic tissue repair and nephro-protection. These findings suggest that pre-placement of S-phase cells in the kidney due to diabetes is critical in mitigating the progression of DCVC-initiated renal injury by upregulation of tissue repair, leading to survival of the DB mice by avoiding acute renal failure.

Expression of an RNAi-resistant SLBP restores proper S-phase progression

Metazoan histone mRNAs are unique in that they lack a 3'-polyadenylated tail, but instead end in a conserved stem loop that is bound by SLBP (stem-loop binding protein). SLBP is required for efficient histone mRNA synthesis and translation. Removal of SLBP by RNA interference causes an increase in the number of cells in S-phase and this effect can be reversed by expressing an exogenous SLBP resistant to the small interfering RNA. Cells with decreased SLBP levels progress slowly through S-phase when released from a double-thymidine block. Thus SLBP is required for efficient DNA replication probably because a decreased ability to assemble chromatin results in a decrease in the rate of DNA replication.

Cell proliferation and gill morphology in anoxic crucian carp

Is DNA replication/cell proliferation in vertebrates possible during anoxia? The oxygen dependence of ribonucleotide reductase (RNR) could lead to a stop in DNA synthesis, thereby making anoxic DNA replication impossible. We have studied this question in an anoxia-tolerant vertebrate, the crucian carp (Carassius carassius), by examining 5'-bromo-2'-deoxyuridine incorporation and proliferating cell nuclear antigen levels in the gills, intestinal crypts, and liver. We exposed crucian carp to 1 and 7 days of anoxia followed by 7 days of reoxygenation. There was a reduced incidence of S-phase cells (from 12.2 to 5.0%) in gills during anoxia, which coincided with a concomitant increase of G(0) cells. Anoxia also decreased the number of S-phase cells in intestine (from 8.1 to 1.8%). No change in the fraction of S-phase cells ( approximately 1%) in liver was found. Thus new S-phase cells after 7 days of anoxia were present in all tissues, revealing a considerable rate of DNA synthesis. Subsequently, the oxygen-dependent subunit of crucian carp RNR (RNRR2) was cloned. We found no differences in amino acids involved in radical generation and availability of the iron center compared with mouse, which could have explained reduced oxygen dependence. Furthermore, the amount of RNRR2 mRNA in gills did not decrease throughout anoxia exposure. These results indicate that crucian carp is able to sustain some cell proliferation in anoxia, possibly because RNRR2 retains its tyrosyl radical in anoxia, and that the replication machinery is still maintained. Although hypoxia triggers a 7.5-fold increase of respiratory surface area in crucian carp, this response was not triggered in anoxia.

Dicoumarol relieves serum withdrawal-induced G 0/1 blockade in HL-60 cells through a superoxide-dependent mechanism

This work was set to study how dicoumarol affects the cell cycle in human myeloid leukemia HL-60 cells. Cells were accumulated in G 0/1 after serum deprivation. However, when cells were treated with 5 μM dicoumarol in serum-free medium, a significant increment in the number of cells in S-phase was observed. Inhibition of G 0/1 blockade was confirmed by the increase of thymidine incorporation, the phosphorylation of retinoblastoma protein, and the promotion of cell growth in long-term treatments in the absence of serum. Dicoumarol treatment increased superoxide levels, but did not affect peroxide. Increase of cellular superoxide was essential for inhibition of G 0/1 blockade, since scavenging this reactive species with a cell-permeable form of SOD and the SOD mimetics 2-amino-3,5-dibromo- N-[ trans-4-hydroxycyclohexyl]benzylamine (ambroxol, 100 μM) and copper[II]diisopropyl salicylate (CuDIPS, 10 μM) completely abolished the effect of dicoumarol. However, N-acetyl-cysteine, overexpression of Bcl-2 or a cell-permeable form of catalase were not effective. 5-Methoxy-1,2-dimethyl-3-[(4-nitrophenol)methyl]-indole-4,7-dione (ES936), a mechanism-based irreversible inhibitor of NAD(P)H:quinone oxidoreductase 1 (NQO1), did not promote S phase entry, and dicoumarol still inhibited G 0/1 blockade in the presence of ES936. We demonstrate that dicoumarol inhibits the normal blockade in G 0/1 in HL-60 cells through a mechanism involving superoxide, but this effect is not dependent solely on the inhibition of the NQO1 catalytic activity. Our results send a precautionary message about use of dicoumarol to elucidate cellular processes involving oxidoreductases.