potential mechanisms involved in resistant phenotype of MCF-7breast carcinoma cells to ionizing radiation induced apoptosis

In the present study, we investigated the mechanisms of apoptosis resistance and the roles of the phosphorylation of BRCA1, p21, the Bax/Bcl-2 protein ratio and cell cycle arrest in IR-induced apoptosis in MCF-7 cells. X-irradiation, in particular at low dose (1 Gy), but not carbon ion irradiation, had a significant antiproliferative effect on the growth of MCF-7 cells. 1 Gy X-irradiation resulted in G1 and G2 phase arrest, but 4 Gy induced a significant G1 block. In contrast, carbon ion irradiation resulted in a significant accumulation in the G2 phase. Concomitant with the phosphorylation of H2AX induced by DNA damage,carbon ion irradiation resulted in an approximately 1.9–2.8-fold increase in the phosphorylation of BRCA1 on serine residue 1524, significantly greater than that detected for X-irradiation. Carbon ion irradiation caused a dramatic increase in p21 expression and drastic decrease in Bax expression compared with X-irradiation. The data implicated that phosphorylation of BRCA1 on serine residue 1524 might,at least partially, induce p21 expression but repress Bax expression. Together, our results suggested that the phosphorylation of BRCA1 at Ser-1524 might contribute to the G2 phase arrest and might be an upstream signal involved in preventing apoptosis signal via upregulation of p21 and downregulation of the Bax/Bcl-2 ratio.

Survivin expressions in human hepatoma HepG2 cells exposed to ionizing radiation of different LET

Survivin is a member of the inhibitors of apoptosis (IAP) protein family that interferes with post-mitochondrial events including activation of caspases. To examine the regulation of survivin expression in response to irradiation with different linear energy transfer (LET), human hepatoma HepG2 cells were irradiated in vitro with X-rays and carbon ions. Cellular sensitivities to low- and high-LET radiation were determined by colony formation. Survivin expression at mRNA and protein level were measured with RT-PCR and Western blot analyses, respectively. Radiation-induced cell cycle arrest and apoptosis were investigated with flow cytometry. We found that low-LET X-rays induced dose-dependent increases in survivin expression. After exposure to high-LET carbon ions, survivin expression gradually increased from 0 to 4 Gy, and then declined at 6 Gy. More pronounced survivin expression, stronger G(2)/M phase arrest was observed after exposure to carbon ions in comparison with X-rays at doses from 0 to 4 Gy. These observations indicate that there is a differential survivin expression in response to different LET radiations and the cycle arrest mechanism may be associated with it. In addition, our data on induction of apoptosis are compatible with the assumption that survivin expression induced by low-LET X-rays radiation may play a critical role in inhibiting apoptosis. However, after irradiation with ions, an anti-apoptotic function of survivin is not evident, possibly because of the serious damage produced by densely ionizing radiation.

γ射线诱导的细胞周期阻滞和适应性反应研究

高剂量电离辐射对健康造成危害，这一点是比较肯定的，而目前人们更关心的是低剂量辐射的健康风险问题。由于缺乏直接的研究数据，低剂量辐射的效应最初是根据线性无闰（LNT）假说推测出来的，推测结果认为任何程度的辐射，无论剂量有多小，对健康都是有害的。但是，LNT假说从提出之日起就受到质疑。近20年的大量实验研究揭示，低剂量辐射可诱导机体和细胞的兴奋效应，低剂量辐射使细胞恶性转化或人群癌症发生率下降，低剂量辐射预先作用减轻继后高剂量照射所造成的有害效应。目前，人们已尝试性地将低剂量辐射效应应用于肿瘤的治疗。本工作以60C0Y射线（0．3oG到min）对肿瘤细胞进行不同的照射：A，假照射，B，scGy照射，c，scGy照射后4h或8h再以3Gy照射，D，3Gy照射。照射后测定细胞周期和克隆存活率。分析了scGyY射线对不同肿瘤细胞细胞周期的影响，scGyY射线诱导的克隆存活适应性反应与细胞周期阻滞适应性反应之间的相关性。最后讨论了本研究结果在肿瘤放射治疗中的潜在应用。本工作结果总结如下：1，scGy丫射线引起hepGZ、HeLa、sMMc-7721和Ho-8910细胞在GZ脑期发生短暂延迟（大致到辐射后4小时），说明细胞周期检查点对损伤非常敏感，很低剂量的辐射即可使之激活。在经过短暂的延迟之后，hePGZ细胞的生长明显加快，照射后24h和48h的相对细胞数分别是对照的124％和216％。结果表明scGy7射线能促进hePGZ细胞的生长。2．3Gyy射线照射后，hepGZ、sMMc-7721和Ho-8910细胞的GZ／M期细胞明显累积，并在照射后12h达到最大值，S期细胞在辐射后6h有一显著累积，此后下降至对照水平。3Gγ照射后的18h内，HeL。细胞的GZ／M期细胞和S期细胞均明显累积。结果表明，3Gyγ射线照射后，hePGZ、sMMc-7721和HO-8910细胞发生GZ／M阻滞，S期短暂延迟，而HeLa细胞的GZ／M期和S期均发生较长时间的延迟，说明HeLa细胞的辐射敏感性和其他三种细胞不一样。这一结果对肿瘤的放射治疗有参考价值。3．在3GyY射线照射之前4h预先照射scGy可使hePGZ和L02细胞在GZ／M期进一步累积，而对HeLa细胞的周期分布没有明显影响，这一结果也说明HeLa细胞的辐射敏感性与其他细胞有差异。4．在3Gyγ射线照射之前8h预先照射scGy可以促进hePGZ细胞通过GZ／M期阻滞。无论两次辐射之间的间隔为4h还是8h，预照射均可诱导hePGZ细胞克降存活适应性反应。这些结果表明，克隆存活适应性反应和细胞周期阻滞适应性反应不是同步出现的，说明克隆存活适应性反应的产生可能并不一定需要细胞从阻滞状态中的恢复，因此，推测这两个方面的适应性反应有一定联系但没有必然相关性。

The experimental research of R-phycoerythrin subunits on cancer treatment: A new photosensitizer in PDT

The mouse tumor cell 5180 and human liver carcinoma cell SMC 7721 cells were first treated with R-PE and its subunits (alpha, beta, gamma subunits), then irradiated with Argon laser (496 nm, 28.8 J/cm(2)). Survival rate was measured by MTT method. In order to compare the phototoxicity in normal cells, the mouse marrow cells were treated with photofrin II and beta-subunit, irradiated with 45 J/cm(2) of light; survival rate was also measured by MTT method. The result showed that R-PE subunits had better PDT effect on s180 cells than R-PE and lower phototoxicity in marrow cells than photofrin II Flow cytometric analysis showed that PDT results in a growth inhibition and a G(0)-G(1) cell cycle arrest in SMC 7721 cells. The tumor cells inhibited by PDT in vivo were morphologically observed by TEM, the tumor cell death was daze to the occlusion of tumor blood vessels and inducement of cell programmed death in nuclei. Therefore, with the advantage in special fluorescence activity, loth molecular weight, good light absorbent character and weak phototoxicity, R-PE subunit is art attractive option for improving the selectivity of PDT.

MYC activity mitigates response to rapamycin in prostate cancer through eukaryotic initiation factor 4E-binding protein 1-mediated inhibition of autophagy.

Loss of PTEN and activation of phosphoinositide 3-kinase are commonly observed in advanced prostate cancer. Inhibition of mammalian target of rapamycin (mTOR), a downstream target of phosphoinositide 3-kinase signaling, results in cell cycle arrest and apoptosis in multiple in vitro and in vivo models of prostate cancer. However, single-agent use of mTOR inhibition has limited clinical success, and the identification of molecular events mitigating tumor response to mTOR inhibition remains a critical question. Here, using genetically engineered human prostate epithelial cells (PrEC), we show that MYC, a frequent target of genetic gain in prostate cancers, abrogates sensitivity to rapamycin by decreasing rapamycin-induced cytostasis and autophagy. Analysis of MYC and the mTOR pathway in human prostate tumors and PrEC showed selective increased expression of eukaryotic initiation factor 4E-binding protein 1 (4EBP1) with gain in MYC copy number or forced MYC expression, respectively. We have also found that MYC binds to regulatory regions of the 4EBP1 gene. Suppression of 4EBP1 expression resulted in resensitization of MYC-expressing PrEC to rapamycin and increased autophagy. Taken together, our findings suggest that MYC expression abrogates sensitivity to rapamycin through increased expression of 4EBP1 and reduced autophagy.

c-Myc is required for maintenance of glioma cancer stem cells.

BACKGROUND: Malignant gliomas rank among the most lethal cancers. Gliomas display a striking cellular heterogeneity with a hierarchy of differentiation states. Recent studies support the existence of cancer stem cells in gliomas that are functionally defined by their capacity for extensive self-renewal and formation of secondary tumors that phenocopy the original tumors. As the c-Myc oncoprotein has recognized roles in normal stem cell biology, we hypothesized that c-Myc may contribute to cancer stem cell biology as these cells share characteristics with normal stem cells. METHODOLOGY/PRINCIPAL FINDINGS: Based on previous methods that we and others have employed, tumor cell populations were enriched or depleted for cancer stem cells using the stem cell marker CD133 (Prominin-1). We characterized c-Myc expression in matched tumor cell populations using real time PCR, immunoblotting, immunofluorescence and flow cytometry. Here we report that c-Myc is highly expressed in glioma cancer stem cells relative to non-stem glioma cells. To interrogate the significance of c-Myc expression in glioma cancer stem cells, we targeted its expression using lentivirally transduced short hairpin RNA (shRNA). Knockdown of c-Myc in glioma cancer stem cells reduced proliferation with concomitant cell cycle arrest in the G(0)/G(1) phase and increased apoptosis. Non-stem glioma cells displayed limited dependence on c-Myc expression for survival and proliferation. Further, glioma cancer stem cells with decreased c-Myc levels failed to form neurospheres in vitro or tumors when xenotransplanted into the brains of immunocompromised mice. CONCLUSIONS/SIGNIFICANCE: These findings support a central role of c-Myc in regulating proliferation and survival of glioma cancer stem cells. Targeting core stem cell pathways may offer improved therapeutic approaches for advanced cancers.

Suppression of DNA-damage checkpoint signaling by Rsk-mediated phosphorylation of Mre11.

Ataxia telangiectasia mutant (ATM) is an S/T-Q-directed kinase that is critical for the cellular response to double-stranded breaks (DSBs) in DNA. Following DNA damage, ATM is activated and recruited by the MRN protein complex [meiotic recombination 11 (Mre11)/DNA repair protein Rad50/Nijmegen breakage syndrome 1 proteins] to sites of DNA damage where ATM phosphorylates multiple substrates to trigger cell-cycle arrest. In cancer cells, this regulation may be faulty, and cell division may proceed even in the presence of damaged DNA. We show here that the ribosomal s6 kinase (Rsk), often elevated in cancers, can suppress DSB-induced ATM activation in both Xenopus egg extracts and human tumor cell lines. In analyzing each step in ATM activation, we have found that Rsk targets loading of MRN complex components onto DNA at DSB sites. Rsk can phosphorylate the Mre11 protein directly at S676 both in vitro and in intact cells and thereby can inhibit the binding of Mre11 to DNA with DSBs. Accordingly, mutation of S676 to Ala can reverse inhibition of the response to DSBs by Rsk. Collectively, these data point to Mre11 as an important locus of Rsk-mediated checkpoint inhibition acting upstream of ATM activation.

Regulation of DNA Double Strand Break Response

To ensure genomic integrity, dividing cells implement multiple checkpoint pathways during the course of the cell cycle. In response to DNA damage, cells may either halt the progression of the cycle (cell cycle arrest) or undergo apoptosis. This choice depends on the extent of damage and the cell's capacity for DNA repair. Cell cycle arrest induced by double-stranded DNA breaks relies on the activation of the ataxia-telangiectasia (ATM) protein kinase, which phosphorylates cell cycle effectors (e.g., Chk2 and p53) to inhibit cell cycle progression. ATM is an S/T-Q directed kinase that is critical for the cellular response to double-stranded DNA breaks. Following DNA damage, ATM is activated and recruited to sites of DNA damage by the MRN protein complex (Mre11-Rad50-Nbs1 proteins) where ATM phosphorylates multiple substrates to trigger a cell cycle arrest. In cancer cells, this regulation may be faulty and cell division may proceed even in the presence of damaged DNA. We show here that the RSK kinase, often elevated in cancers, can suppress DSB-induced ATM activation in both Xenopus egg extracts and human tumor cell lines. In analyzing each step in ATM activation, we have found that RSK disrupts the binding of the MRN complex to DSB DNA. RSK can directly phosphorylate the Mre11 protein at Ser 676 both in vitro and in intact cells and can thereby inhibit loading of Mre11 onto DSB DNA. Accordingly, mutation of Ser 676 to Ala can reverse inhibition of the DSB response by RSK. Collectively, these data point to Mre11 as an important locus of RSK-mediated checkpoint inhibition acting upstream of ATM activation.

The phosphorylation of Mre11 on Ser 676 is antagonized by phosphatases. Here, we screened for phosphatases that target this site and identified PP5 as a candidate. This finding is consistent with the fact that PP5 is required for the ATM-mediated DNA damage response, indicating that PP5 may promote DSB-induced, ATM-dependent DNA damage response by targeting Mre11 upstream of ATM.

HDAC inhibitors cause site-specific chromatin remodeling at PU.1-bound enhancers in K562 cells

BACKGROUND: Small molecule inhibitors of histone deacetylases (HDACi) hold promise as anticancer agents for particular malignancies. However, clinical use is often confounded by toxicity, perhaps due to indiscriminate hyperacetylation of cellular proteins. Therefore, elucidating the mechanisms by which HDACi trigger differentiation, cell cycle arrest, or apoptosis of cancer cells could inform development of more targeted therapies. We used the myelogenous leukemia line K562 as a model of HDACi-induced differentiation to investigate chromatin accessibility (DNase-seq) and expression (RNA-seq) changes associated with this process. RESULTS: We identified several thousand specific regulatory elements [~10 % of total DNase I-hypersensitive (DHS) sites] that become significantly more or less accessible with sodium butyrate or suberanilohydroxamic acid treatment. Most of the differential DHS sites display hallmarks of enhancers, including being enriched for non-promoter regions, associating with nearby gene expression changes, and increasing luciferase reporter expression in K562 cells. Differential DHS sites were enriched for key hematopoietic lineage transcription factor motifs, including SPI1 (PU.1), a known pioneer factor. We found PU.1 increases binding at opened DHS sites with HDACi treatment by ChIP-seq, but PU.1 knockdown by shRNA fails to block the chromatin accessibility and expression changes. A machine-learning approach indicates H3K27me3 initially marks PU.1-bound sites that open with HDACi treatment, suggesting these sites are epigenetically poised. CONCLUSIONS: We find HDACi treatment of K562 cells results in site-specific chromatin remodeling at epigenetically poised regulatory elements. PU.1 shows evidence of a pioneer role in this process by marking poised enhancers but is not required for transcriptional activation.

p53 Regulates the Formation of Lamellipodia and Circular Dorsal Ruffles Through Caldesmon and PTEN

Vascular smooth muscle cell migration is a significant contributor to many aspects of heart disease, and specifically atherosclerosis. Tissue damage in the arteries can result in the formation of a fatty streak. Smooth muscle cells (SMC) can then migrate to this site to form a fibrous cap, stabilizing the fatty plaque. Since cardiovascular disease is the leading cause of death in developed countries, this function of SMC is an essential area of study. The formation of lamellipodia and circular dorsal ruffles were studied in this project as indicators that cell migration is occurring. The roles of the proteins p53, Rac, caldesmon and PTEN were investigated with regards to these actin-based structures. The tumour suppressor p53 is often reported to cause apoptosis, senescence or cell cycle arrest when stress is placed on a cell, but has recently been shown to regulate cell migration as well. It was determined in this project that p53 could inhibit the formation of both lamellipodia and circular dorsal ruffles. It was also shown that this could occur directly through an inhibition of the GTPase Rac. Previous studies have shown that p53 can upregulate caldesmon, a protein which is known to bind to and stabilize actin filaments while inhibiting Arp2/3-mediated branching. It was confirmed that p53 could upregulate caldesmon, and that caldesmon could inhibit the formation of lamellipodia and circular dorsal ruffles. The phosphorylation of caldesmon by p21-associated kinase (PAK) or extracellular signal-related kinase (Erk) was shown to effectively reverse the ability of caldesmon to inhibit these structures. The role of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) was also studied with regards to this signalling pathway. PTEN was shown to inhibit lamellipodia and circular dorsal ruffles through its lipid phosphatase activity. It was concluded that p53 can inhibit the formation of lamellipodia and circular dorsal ruffles in vascular SMC, and that this occurs through Rac, caldesmon and PTEN.

The Role of Thymidylate Synthase Induction in Modulating p53-regulated Gene Expression in Response to 5-Fluorouracil and Antifolates

Thymidylate synthase (TS) is a critical target for chemotherapeutic agents such as 5-fluorouracil (5-FU) and antifolates such as tomudex (TDX),multitargeted antifolate, and ZD9331. Using the MCF-7 breast cancer line, we have developed p53 wild-type (M7TS90) and null (M7TS90-E6) isogenic lines with inducible TS expression (approximately 6-fold induction compared with control after 48 h). In the M7TS90 line, inducible TS expression resulted in a moderate approximately 3-fold increase in 5-FU IC-50(72 h) dose and a dramatic >20-fold increase in the IC-50(72 h) doses of TDX, multitargeted antifolate, and ZD9331. S-phase cell cycle arrest and apoptosis induced by the antifolates were abrogated by TS induction. In contrast, cell cycle arrest and apoptosis induced by 5-FU was unaffected by TS expression levels. Inactivation of p53 significantly increased resistance to 5-FU and the antifolates with IC-50(72 h) doses for 5-FU and TDX of >100 and >10 microM, respectively, in the M7TS90-E6 cell line. Furthermore, p53 inactivation completely abrogated the cell cycle arrest and apoptosis induced by 5-FU. The antifolates induced S-phase arrest in the p53 null cell line; however, the induction of apoptosis by these agents was significantly reduced compared with p53 wild-type cells. Both inducible TS expression and the addition of exogenous thymidine (10 microM) blocked p53 and p21 induction by the antifolates but not by 5-FU in the M7TS90 cell line. Similarly, inducible TS expression and exogenous thymidine abrogated antifolate but not 5-FU-mediated up-regulation of Fas/CD95 in M7TS90 cells. Our results indicate that in M7TS90 cells, inducible TS expression modulates p53 and p53 target gene expression in response to TS-targeted antifolate therapies but not to 5-FU.

Role played by BRCA1 in regulating the cellular response to stress

BRCA1 (breast-cancer susceptibility gene 1) is a tumour suppressor gene that is mutated in the germline of women with a genetic predisposition to breast and ovarian cancer. In this review, we examine the role played by BRCA1 in mediating the cellular response to stress. We review the role played by BRCA1 in detecting and signalling the presence of DNA damage, particularly double-strand DNA breaks, and look at the evidence to support a role for BRCA1 in regulating stress response pathways such as the c-Jun N-terminal kinase/stress-activated protein kinase pathway. in addition, we examine the role played by BRCA1 in mediating both cell-cycle arrest and apoptosis following different types of cellular insult, and how this may be modulated by the presence or absence of associated proteins such as p53. Finally, we explore the possibility that many of the functions associated with BRCA1 may be based on transcriptional regulation of key downstream genes that have been implicated in the regulation of these specific cellular pathways.

Estrogen Receptor-beta Affects the Prognosis of Human Malignant Mesothelioma

Malignant pleural mesothelioma is an asbestos-related neoplasm with poor prognosis, refractory to current therapies, the incidence of which is expected to increase in the next decades. Female gender was identified as a positive prognostic factor among other clinical and biological prognostic markers for malignant mesothelioma, yet a role of estrogen receptors (ERs) has not been studied. Our goal was to investigate ERs expression in malignant mesothelioma and to assess whether their expression correlates with prognosis. Immunohistochemical analysis revealed intense nuclear ER beta staining in normal pleura that was reduced in tumor tissues. Conversely, neither tumors nor normal pleura stained positive for ER alpha. Multivariate analysis of 78 malignant mesothelioma patients with pathologic stage, histologic type, therapy, sex, and age at diagnosis indicated that FRO expression is an independent prognostic factor of better survival. Moreover, studies in vitro confirmed that treatment with 17 beta-estradiol led to an ER beta-mediated inhibition of malignant mesothelioma cell proliferation as well as p21(CIP1) and p27(KIP1) up-regulation. Consistently cell growth was suppressed by ER beta overexpression, causing a G(2)-M-phase cell cycle arrest, paralleled by cyclin B1 and survivin down-regulation. Our data support the notion that ER beta acting as a tumor suppressor is of high potential relevance to prediction of disease progression and to therapeutic response of malignant mesothelioma patients. [Cancer Res 2009;69(11):4598-604]

Cdk1 participates in BRCA1-dependent S phase checkpoint control in response to DNA damage.

Cdk2 and cdk1 are individually dispensable for cell-cycle progression in cancer cell lines because they are able to compensate for one another. However, shRNA-mediated depletion of cdk1 alone or small molecule cdk1 inhibition abrogated S phase cell-cycle arrest and the phosphorylation of a subset of ATR/ATM targets after DNA damage. Loss of DNA damage-induced checkpoint control was caused by a reduction in formation of BRCA1-containing foci. Mutation of BRCA1 at S1497 and S1189/S1191 resulted in loss of cdk1-mediated phosphorylation and also compromised formation of BRCA1-containing foci. Abrogation of checkpoint control after cdk1 depletion or inhibition in non-small-cell lung cancer cells sensitized them to DNA-damaging agents. Conversely, reduced cdk1 activity caused more potent G2/M arrest in nontransformed cells and antagonized the response to subsequent DNA damage. Cdk1 inhibition may therefore selectively sensitize BRCA1-proficient cancer cells to DNA-damaging treatments by disrupting BRCA1 function.

The Novel Tubulin-Targeting Agent Pyrrolo-1,5-Benzoxazepine-15 Induces Apoptosis in Poor Prognostic Subgroups of Chronic Lymphocytic Leukemia

Pyrrolo-1,5-benzoxazepine-15 (PBOX-15) is a novel microtubule depolymerization agent that induces cell cycle arrest and subsequent apoptosis in a number of cancer cell lines. Chronic lymphocytic leukemia (CLL) is characterized by clonal expansion of predominately nonproliferating mature B cells. Here, we present data suggesting PBOX-15 is a potential therapeutic agent for CLL. We show activity of PBOX-15 in samples taken from a cohort of CLL patients (n = 55) representing both high-risk and low-risk disease. PBOX-15 exhibited cytotoxicity in CLL cells (n = 19) in a dose-dependent manner, with mean IC(50) of 0.55 mu mol/L. PBOX-15 significantly induced apoptosis in CLL cells (n = 46) including cells with poor prognostic markers: unmutated IgV(II) genes, CD38 and zeta-associated protein 70 (ZAP-70) expression, and fludarabine-resistant cells with chromosomal deletions in 17p. In addition, PBOX-15 was more potent than fludarabine in inducing apoptosis in fludarabine-sensitive cells. Pharmacologic inhibition and small interfering RNA knockdown of caspase-8 significantly inhibited PBOX-15-induced apoptosis. Pharmacologic inhibition of c-jun NH(2)-terminal kinase inhibited PBOX-15-induced apoptosis in mutated IgV(II) and ZAP-70(-) CLL cells but not in unmutated IgV(II) and ZAP-70(+) cells. PBOX-15 exhibited selective cytotoxicity in CLL cells compared with normal hematopoietic cells. Our data suggest that PBOX-15 represents a novel class of agents that are toxic toward both high-risk and low-risk CLL cells. The need for novel treatments is acute in CLL, especially for the subgroup of patients with poor clinical outcome and drug-resistant disease. This study identifies a novel agent with significant clinical potential.