The role of apoptosis in 2',2'-difluoro-2'-deoxycytidine (gemcitabine)-mediated radiosensitization.

The nucleoside analogue Gemcitabine [2',2'-difluoro-2'-deoxycytidine (dFdCyd)] is active against a wide variety of solid tumors and is a potent radiation sensitizer. Because apoptosis has been shown to be an important mechanism of cell death for many cancers, we wished to investigate the role of apoptosis in dFdCyd-mediated radiosensitization. We evaluated HT29 colon cancer cells, UMSCC-6 head and neck cancer cells, and A549 lung cancer cells, which differ substantially in the ability to undergo radiation-induced apoptosis. We hypothesized that if dFdCyd produced radiosensitization by potentiating preexisting death pathways, then only the apoptotic-prone HT29 cells would show a substantial increase in apoptosis when treated with the combination of dFdCyd and radiation and that UMSCC-6 cells and A549 cells would be radiosensitized through nonapoptotic mechanisms. We found that the radiosensitization of HT29 cells (enhancement ratio, 1.81 +/- 0.16) was accompanied by an increase in apoptosis and by caspase activation and that inhibition of this activation by the caspase inhibitor Z-Asp-Glu-Val-Asp-fluoromethylketone (DEVD) significantly decreased radiosensitization (to 1.36 +/- 0.24; P < 0.05). In contrast, UMSCC-6 cells and A549 cells were modestly radiosensitized (enhancement ratio, 1.47 +/- 0.24 and 1.31 +/- 0.04, respectively) via a nonapoptotic mechanism. These findings suggest that although apoptosis can contribute significantly to dFdCyd-mediated radiosensitization, the role of apoptosis in dFdCyd-mediated radiosensitization depends on the cell line rather than representing a general property of the drug.     

Evaluation of the antitumor activity of gemcitabine (2',2'-difluoro-2'-deoxycytidine)

A new pyrimidine antimetabolite, 2',2'-difluorodeoxycytidine, Gemcitabine (LY188011, dFdCyd) has been synthesized and evaluated in experimental tumor models. dFdCyd is a very potent and specific deoxycytidine analogue. The concentration required for 50% inhibition of growth is 1 ng/ml in the CCRF-CEM human leukemia cell culture assay. Concurrent addition of deoxycytidine to the cell culture system provides about a 1000-fold decrease in biological activity. The inhibition of growth of human leukemia cells in culture led to the in vivo evaluation of this compound as a potential oncolytic agent. Maximal activity in vivo was seen with dFdCyd when administered on an every third day schedule. 1-beta-D-Arabinofuranosylcytosine, administered on a daily for 10-day schedule, was directly compared to dFdCyd in this evaluation. dFdCyd demonstrated good to excellent antitumor activity in eight of the eight murine tumor models evaluated. 1-beta-D-Arabinofuranosylcytosine was substantially less active or had no activity in these same tumor models. This in vivo activity against murine solid tumors supports the conclusion that dFdCyd is an excellent candidate for clinical trials in the treatment of cancer.     

Radiosensitization produced in vivo by once- vs. twice-weekly 2'2'-difluoro-2'-deoxycytidine (gemcitabine)

PURPOSE: Gemcitabine (2'2'-difluoro-2'-deoxycytidine, dFdCyd) is a potent radiosensitizer of rodent and human tumor cells. Our Phase I clinical trial using once-weekly dFdCyd as a radiosensitizer in the treatment of patients with Stage IV squamous cell head and neck cancer has produced a high rate of tumor response and significant normal mucosal toxicity. These findings raised the question of whether we are using dFdCyd in the optimal dose and schedule. In vitro studies suggest that twice-weekly dFdCyd has the potential to be more effective than once-weekly dFdCyd when administered in combination with radiation (RT) given 5 days per week. Therefore, we have used a mouse model to assess whether the therapeutic ratio of combined modality therapy may be improved by using a twice-weekly drug regimen. We asked two questions: 1) Does a once-weekly or twice-weekly dFdCyd regimen cause more normal tissue radiosensitization? 2) Does a once-weekly or twice-weekly dFdCyd + RT regimen produce a better therapeutic index? METHODS AND MATERIALS: To assess normal tissue toxicity, C3H mice underwent mouth (60)Co RT (27.5 Gy in 5 daily fractions) +/- dFdCyd delivered intraperitoneally (IP) either once or twice weekly 6 hours prior to irradiation. Acute lip reactions were quantified according to a standard scoring system, and weight loss was measured. We measured tumor control using squamous cell carcinoma (SCC) VII murine squamous cell flank tumors (50-125 mm(3)) treated with the same regimens used in the mouth irradiation model. RESULTS: We found that dFdCyd delivered 800 mg/kg once weekly or 150 mg/kg twice weekly caused similar (and maximal tolerable) weight loss; therefore these regimens were chosen to test which schedule produced more acute lip radiosensitization. Twice-weekly dFdCyd + RT was somewhat more toxic by weight loss (800 mg/kg once weekly: 11.9%; 150 mg/kg twice weekly: 17.7%; p = 0.09). To assess therapeutic index, we treated SCC VII flank tumors with RT combined with isotoxic drug/RT regimens (dFdCyd 800 mg/kg once weekly or 100 mg/kg twice weekly). Tumors treated with twice-weekly dFdCyd + RT were significantly smaller than tumors treated with once-weekly drug + RT at 28 days from the start of treatment (p < 0.03). CONCLUSIONS: These findings demonstrate that equitoxic once- versus twice-weekly dFdCyd regimens cause differing levels of oral mucosal radiosensitization. This would suggest that each radiation-dFdCyd schedule will require its own dFdCyd dose escalation trial (which cannot be determined by the maximum tolerated dose (MTD) for dFdCyd alone using that schedule). In addition, our findings suggest that for head and neck cancers twice-weekly dFdCyd may have a higher therapeutic index compared with once-weekly dFdCyd when combined with daily RT.     

Gemcitabine (2',2'-difluoro-2'-deoxycytidine), an antimetabolite that poisons topoisomerase I.

PURPOSE: Gemcitabine-containing regimens are among standard therapies for the treatment of advanced non-small cell lung,pancreatic, or bladder cancers. Gemcitabine is a nucleoside analogue and its cytotoxicity is correlated with incorporation into genomic DNA and concomitant inhibition of DNA synthesis. However, it is still unclear by which mechanism(s) gemcitabine incorporation leads to cell death. EXPERIMENTAL DESIGN: We used purified oligodeoxynucleotides to study the effects of gemcitabine incorporation on topoisomerase I (top1) activity and tested the role of top1 poisoning in gemcitabine-induced cytotoxicity in cancer cells. RESULTS: We found that top1-mediated DNA cleavage was enhanced when gemcitabine was incorporated immediately 3' from a top1 cleavage site on the nonscissile strand. This position-specific enhancement was attributable to an increased DNA cleavage by top1 and was likely to have resulted from a combination of gemcitabine-induced conformational and electrostatic effects. Gemcitabine also enhanced camptothecin-induced cleavage complexes. We also detected top1 cleavage complexes in human leukemia CEM cells treated with gemcitabine and a 5-fold resistance of P388/CPT45 top1-deficient cells to gemcitabine, indicating that poisoning of top1 can contribute to the antitumor activity of gemcitabine. CONCLUSIONS: The present results extend our recent finding that incorporation of 1-beta-D-arabinofuranosylcytosine into DNA can induce top1 cleavage complexes [P. Pourquier et al. Proc. Natl. Acad. Sci. USA, 97: 1885-1890, 2000]. The enhancement of camptothecin-induced top1 cleavage complexes may, at least in part, contribute to the synergistic or additive effects of gemcitabine in combination with topotecan and irinotecan in human breast or lung cancer cells.     

Modulation of gemcitabine (2',2'-difluoro-2'-deoxycytidine) pharmacokinetics, metabolism, and bioavailability in mice by 3,4,5,6-tetrahydrouridine.

PURPOSE: In vivo, 2',2'-difluoro-2'-deoxycytidine (dFdC) is rapidly inactivated by gut and liver cytidine deaminase (CD) to 2',2'-difluoro-2'-deoxyuridine (dFdU). Consequently, dFdC has poor oral bioavailability and is administered i.v., with associated costs and limitations in administration schedules. 3,4,5,6-Tetrahydrouridine (THU) is a potent CD inhibitor with a 20% oral bioavailability. We investigated the ability of THU to decrease elimination and first-pass effect by CD, thereby enabling oral dosing of dFdC. EXPERIMENTAL DESIGN: A liquid chromatography-tandem mass spectrometry assay was developed for plasma dFdC and dFdU. Mice were dosed with 100 mg/kg dFdC i.v. or orally with or without 100 mg/kg THU i.v. or orally. At specified times between 5 and 1,440 min, mice (n = 3) were euthanized. dFdC, dFdU, and THU concentrations were quantitated in plasma and urine. RESULTS: THU i.v. and orally produced concentrations >4 microg/mL for 3 and 2 h, respectively, whereas concentrations of >1 microg/mL have been associated with near-complete inhibition of CD in vitro. THU i.v. decreased plasma dFdU concentrations but had no effect on dFdC plasma area under the plasma concentration versus time curve after i.v. dFdC dosing. Both THU i.v. and orally substantially increased oral bioavailability of dFdC. Absorption of dFdC orally was 59%, but only 10% passed liver and gut CD and eventually reached the systemic circulation. Coadministration of THU orally increased dFdC oral bioavailability from 10% to 40%. CONCLUSIONS: Coadministration of THU enables oral dosing of dFdC and warrants clinical testing. Oral dFdC treatment would be easier and cheaper, potentially prolong dFdC exposure, and enable exploration of administration schedules considered impractical by the i.v. route.     

抑制ATM表达致鼻咽癌细胞CNE1辐射增敏的细胞周期阻滞研究

背景与目的:细胞周期调控是决定细胞辐射敏感性的决定性因素之一。共济失调毛细血管扩张症突变基因(ataxia-telangiectasia mutant,ATM)功能与细胞DNA损伤修复、细胞周期检查点调控密切相关。我们前期研究通过反义RNA抑制ATM基因表达可增加鼻咽癌细胞系CNE1辐射敏感性,本研究拟探讨其辐射增敏的细胞周期阻滞调控机制。方法:ATM反义组细胞CNE1/pDOR-atm及对照组细胞CNE1/pDOR经2Gy、5GyX线照射后不同时间点(1h、4h、8h、24h、48h)收获,应用流式细胞仪(flowcytometer,FCM)检测各细胞周期百分比及凋亡率。结果:两组细胞X线照射后均未出现明显G1期阻滞和细胞凋亡,但分别在照射后1h、4h、8h出现明显S期阻滞,24h、48h出现明显G2期阻滞,其中反义组S期细胞百分率总均数水平低于对照组(P<0.05),而G2/M期细胞百分率总均数水平高于对照组(P<0.05)。结论:反义RNA抑制ATM表达致CNE1辐射增敏的细胞周期调控机制可能与减少S期细胞比例,增加G2/M期细胞比例有关,与G1期阻滞和细胞凋亡的调控无关。     

Effects of 5-aza-2'-deoxycytidine on survival and cell cycle progression of L1210 leukemia cells

The in-vitro effects of the antileukemic agent 5-aza-2'-deoxycytidine (5-aza-dCyd), on DNA synthesis, growth, cloning in agar, and cell cycle traverse of L1210 leukemia cells were studied. 5-Aza-dCyd at 0.1 microgram/ml for 10 hr (cytotoxic concentration) did not inhibit DNA synthesis but produced a very potent growth inhibition, and changed markedly the DNA flow cytometric histograms. A 5-h continuous exposure to the drug at concentrations ranging from 0.1 to 10 micrograms/ml caused an accumulation of cells in the S portion of the DNA histograms indicating a slowing of the progression of cells in the S phase. A longer exposure time (10 h) at the same concentrations led to a bimodal DNA distribution (peaks at G1 and G2-M) and a depletion of the S phase. When the exposure time to 5-aza-dCyd (0.1 microgram/ml) was extended to 15 and 20 h, there was a decrease in the G2-M peak and an augmentation of the G1 peak. To determine if 5-aza-dCyd produced a block in cell cycle progression, L1210 cells were treated for 10 h with colcemid and 5-aza-dCyd simultaneously for 10 h. Colcemid alone, or colcemid in combination with 5-aza-dCyd produced an accumulation of cells under a single G2-M peak. This indicates that 5-aza-dCyd did not block the progression of L1210 cells through S phase, but only produced a slowing down of this event. These results, indicating that 5-aza-dCyd does not block cell cycle progression and that its cytotoxic action is not self-limiting, are of importance for designing future clinical trials.     

Tumor uptake and elimination of 2',2'-difluoro-2'-deoxycytidine (gemcitabine) after deoxycytidine kinase gene transfer: correlation with in vivo tumor response.

PURPOSE: We hypothesized that tumor uptake and elimination of 2',2'-difluoro-2'-deoxycytidine/2',2'-difluoro-2'-deoxycytidine 5'-triphosphate (dFdCyd/dFdCTP) would be altered after dCK gene transfer and that this change would result in an enhanced cytotoxic effect. To test this hypothesis, we examined dFdCyd/dFdCTP uptake and clearance in HT-29 human colon carcinoma xenografts in nude mice by high-performance liquid chromatography (HPLC) and fluorine-19 magnetic resonance spectroscopy (F-19 MRS). EXPERIMENTAL DESIGN: HT-29 tumors were grown from cells infected with either the retroviral vector alone (LNPO-LacZ) or vector containing the dCK gene (LNPO-dCK). HPLC and F-19 MRS analyses were performed after a single 160 mg/kg i.p. injection of dFdCyd. Tumor response was determined in animals receiving a similar dosing schedule of dFdCyd. RESULTS: HPLC experiments revealed an increased tumor accumulation of dFdCTP in xenografts overexpressing dCK compared with wild-type controls (P < or = 0.05). dFdCTP in the dCK-infected tumors was easily identified at 24 h postinjection. Conversely, no dFdCTP could be detected in the control xenografts 14 h postinjection. Subsequent F-19 MRS experiments confirmed an altered uptake, revealing a 2.5-fold greater accumulation of dFdCyd/dFdCTP in the dCK xenografts. Whereas a modest tumor growth delay was observed in the wild-type tumors receiving dFdCyd, dCK xenografts demonstrated a marked tumor growth delay following treatment (P < or = 0.05). CONCLUSIONS: These data support the hypothesis that increased expression of dCK cDNA in HT-29 xenografts results in an enhanced dFdCTP accumulation and prolonged elimination kinetics, and ultimately a potentiated in vivo tumor response to dFdCyd. Related to these effects, changes in the overall tumor metabolism of dFdCyd/dFdCTP was detectable by noninvasive F-19 MRS. These data are relevant to future preclinical and clinical studies evaluating dCK gene transfer and dFdCyd therapy.     

Interleukin-2 expression in human carcinoma cell lines and its role in cell cycle progression

Human carcinomas were shown to express mRNA and protein for IL-2R alpha, beta and gamma chains. Recently, human carcinomas were also shown to constitutively express protein and mRNA for IL-2 in vivo and in vitro. Here we report that the expression levels of cytoplasmic IL-2 as well as IL-2Rbeta- and gamma-chain in human carcinoma cells change during the cell cycle progression. Carcinoma cells synchronized in the G2/M phase of the cell cycle expressed significantly more intracytoplasmic IL-2 as well as IL-2Rbeta and gamma proteins than tumor cells in the G0/G1 phase. The level of mRNA for IL-2 was 5-10-fold higher in the M phase than in the G0/G1-phase, as shown by quantitative competitive RT-PCR. Expression of the cyclin-dependent kinase (CDK) inhibitor p27kip1 in these carcinoma cells was found to be high in the G0/G1 phase, nearly absent in the S phase, and it increased again in the G2/M phase of the cell cycle. In synchronized cells, the decrease in p27 expression coincided with high levels of expression of IL-2. Using the IL-2 specific antisense oligonucleotide to block synthesis of endogenous IL-2 in tumor cells, we observed increased levels of p27 as well as p21. The antisense oligonucleotides specific for p27 or p21 blocked expression of these proteins but not of IL-2. Thus, endogenous IL-2 is important in regulating expression of p27 as well as p21 and, therefore, in controlling cell cycle progression of tumor cells, while its own expression remains independent of the CDK inhibitors.     

Cell killing, radiosensitization and cell cycle redistribution induced by chronic hypoxia

Some of the biological changes associated with extreme hypoxia at 37 degrees C (less than 10 ppm pO2) were examined in Chinese hamster V79 cells. Specifically, extreme hypoxia caused an initial decrease in plating efficiency to 55% in 4 hr after the onset of hypoxia. Beyond this time, the decline in plating efficiency was more gradual reaching 35% of control at 20 hr. Flow microfluorimetry (FMF) studies, in which cells are sorted on the basis of DNA content and then assayed for viability, demonstrated that mid S phase cells were most sensitive to chronic hypoxia, with surviving fraction equal to 2.5% at 20 hr. Furthermore, the viability of G1 and G2/M cells, after 20 hr of hypoxic storage, was also reduced to 20 and 7.6%, respectively. Hypoxia also caused alterations in the cell cycle distribution of initially asynchronous cells, as determined by dual parameter FMF measurements of both cellular DNA content and incorporated BudR. In particular, G2/M cells completed mitosis, while G1 cells showed little or no movement. Lastly, cells stored in chronic hypoxia displayed an enhanced radiosensitivity when compared to acutely hypoxic cells. Possible reasons for these observations are discussed.     

Kinetics of mouse jejunum radiosensitization by 2',2'-difluorodeoxycytidine (gemcitabine) and its relationship with pharmacodynamics of DNA synthesis inhibition and cell cycle redistribution in crypt cells.

Gemcitabine (dFdC), a deoxycitidine nucleoside analogue, inhibits DNA synthesis and repair of radiation-induced chromosome breaks in vitro, radiosensitizes various human and mouse cells in vitro and shows clinical activity in several tumours. Limited data are however available on the effect of dFdC on normal tissue radiotolerance and on factors associated with dFdC''s radiosensitization in vivo. The purpose of this study was to determine the effect of dFdC on mouse jejunum radiosensitization and to investigate the kinetics of DNA synthesis inhibition and cell cycle redistribution in the jejunal crypts as surrogates of radiosensitization in vivo. For assessment of jejunum tolerance, the mice were irradiated on the whole body with 60Co gamma rays (3.5-18 Gy single dose) with or without prior administration of dFdC (150 mg kg-1). Jejunum tolerance was evaluated by the number of regenerated crypts per circumference at 86 h after irradiation. For pharmacodynamic studies, dFdC (150 or 600 mg kg-1) was given i.p. and jejunum was harvested at various times (0-48 h), preceded by a pulse BrdUrd labelling. Labelled cells were detected by immunohistochemistry on paraffin-embedded sections. DNA synthesis was inhibited within 3 h after dFdC administration. After an early wave of apoptosis (3-6 h), DNA synthesis recovered by 6 h, and crypt cells became synchronized. At 48 h, the labelling index returned almost to background level. At a level of 40 regenerated crypts, radiosensitization was observed for a 3 h time interval (dose modification factor of 1.3) and was associated with DNA synthesis inhibition, whereas a slight radioprotection was observed for a 48-h time interval (dose modification factor of 0.9) when DNA synthesis has reinitiated. In conclusion, dFdC altered the radioresponse of the mouse jejunum in a schedule-dependent fashion. Our data tend to support the hypothesis that DNA synthesis inhibition and cell cycle redistribution are surrogates for radiosensitization. More data points are however required before a definite conclusion can be drawn.     

Regulation of normal cell cycle progression by flavin-containing oxidases

Mechanisms underlying the role of reactive oxygen species (ROS) generated by flavin-containing oxidases in regulating cell cycle progression were examined in human and rodent fibroblasts. Incubation of confluent cell cultures with nontoxic/nonclastogenic concentrations of the flavoprotein inhibitor, diphenyleneiodonium (DPI), reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase activity and basal ROS levels, but increased proteolysis of cyclin D1, p21(Waf1) and phospho-p38(MAPK). When these cells were allowed to proliferate by subculture in DPI-free medium, an extensive G(1) delay was observed with concomitant activation of p53/p21(Waf1) signaling and reduced phosphorylation of mitogen-activated kinases. Compensation for decreased oxidant generation by simultaneous exposure to DPI and nontoxic doses of the ROS generators, gamma-radiation or t-butyl-hydroperoxide, attenuated the G(1) delay. Whereas the DPI-induced G(1) checkpoint was completely dependent on PHOX91, ATM and WAF1, it was only partially dependent on P53. Interestingly, G(1) to S progression was not affected when another flavin-containing enzyme, nitric oxide synthase, was inhibited nor was it associated with changes in mitochondrial membrane potential. Proliferating cells treated with DPI also experienced a significant but attenuated delay in G(2). We propose that ATM performs a critical function in mediating normal cellular proliferation that is regulated by nonphagocytic NAD(P)H oxidase enzymes activity, which may serve as a novel target for arresting cancer cells in G(1).     

Inhibition of cell cycle progression on HepG2 cells by hypsiziprenol A9, isolated from Hypsizigus marmoreus

Antiproliferative activities of fractions of Hypsizigus marmoreus were examined using HepG2 cells in vitro. The methanol extract of H. marmoreus markedly induced antiproliferative activity, and an active compound from this mushroom was identified as hypsiziprenol A9. Hypsiziprenol A9 inhibited cell proliferation in a time- and concentration-dependent manner by up to 80% on HepG2 cells by inducing arrest of the G1 phase. Further investigation revealed that hypsiziprenol A9 decreased expression of phosphorylated retinoblastoma protein (ppRb), cyclin D1, and cyclin E in a dose-dependent manner. These results suggest that hypsiziprenol A9 can inhibit the growth of HepG2 cells through inducing G1 phase cell cycle arrest due to the inhibition of pRb phosphorylation.     

The role of EZH2 in tumour progression

Accumulated evidence shows that EZH2 is deregulated in a wide range of cancer types, and it has a crucial role in stem cell maintenance and tumour development. Therefore, blocking EZH2 expression or activity may represent a promising strategy for anticancer treatment. In this review, we address the current understanding of the mechanisms underlying EZH2 regulation alongside the function of EZH2 gene targets that are involved in cancer progression. Finally, we will describe cancer therapies that target EZH2 or its downstream cascades, which could potentially reverse the oncogenic and stemness properties of the tumour cells to suppress cancer progression and recurrence.     

CXCR2在肿瘤进展中的作用

CXCR2与人体免疫关系密切,多项研究表明CXCR2及其配体在结肠癌、乳腺癌、肝癌、肾细胞癌、黑色素瘤、胰腺癌、卵巢癌等多种肿瘤细胞中呈高表达,在促进肿瘤生长、转移、血管生成等肿瘤进展过程中发挥重要作用。CXCR2在多种肿瘤的诊断以及预后的判断上具有重要价值。包括CXCR2拮抗剂SB225002在内的多种趋化因子拮抗剂在多项研究中都表现出了抑制血管生成及抑制肿瘤细胞生长、转移等作用。文章对CXCR2及其配体在肿瘤进展中的作用进行综述,为肿瘤的靶向治疗提供新的思路。     

Bcl-2 expression delays hepatocyte cell cycle progression during liver regeneration

Bcl-2 is the prototype of a family of genes that prevent apoptosis. However, several reports indicate that Bcl-2 may also act as a cell cycle modulator. In several human tumors, Bcl-2 expression correlates with a more favorable prognosis and lower tumor proliferative activity. We have shown that Bcl-2 expression delays liver tumor development in transgenic mice even when the gene is turned on shortly before the time of tumor development. We hypothesized that Bcl-2 may delay liver tumorigenesis by interfering with hepatocyte proliferation. To test whether Bcl-2 expression may act on hepatocyte replication we studied liver regeneration in Bcl-2 transgenic mice and wild-type littermates. DNA replication was delayed by approximately 8 h in Bcl-2 transgenic mice compared to the timing of the response in wild-type littermates. Cyclin D expression showed no alterations in the regenerating liver of Bcl-2 transgenic mice. In contrast, there was a delay in the expression of p107, cyclin E and in the activity of cyclin E/cdk 2 activity. These results show that Bcl-2 expression delays cell cycle progression in hepatocytes and suggests that it acts at a step involving cyclin E and p107.