NSCLC cells demonstrate differential mode of cell death in response to the combined treatment of radiation and a DNA-PKcs inhibitor

The current standard of care for lung cancer consists of concurrent chemotherapy and radiation. Several studies have shown that the DNA-PKcs inhibitor NU7441 is a highly potent radiosensitizer, however, the mechanism of NU7441''s anti-proliferation effect has not been fully elucidated. In this study, the combined effect of NU7441 and ionizing radiation (IR) in a panel of non-small cell lung cancer cell lines (A549, H460 and H1299) has been investigated. We found that NU7441 significantly enhances the effect of IR in all cell lines. The notable findings in response to this combined treatment are (i) prolonged delay in IR-induced DNA DSB repair, (ii) induced robust G2/M checkpoint, (iii) increased aberrant mitosis followed by mitotic catastrophe specifically in H1299, (iv) dramatically induced autophagy in A549 and (v) IR-induced senescence specifically in H460. H1299 cells show greater G2 checkpoint adaptation after combined treatment, which can be attributed to higher expression level of Plk1 compared to A549 and H460. The enhanced autophagy after NU7441 treatment in A549 is possibly due to the higher endogenous expression of pS6K compared to H1299 and H460 cells. In conclusion, choice of cell death pathway is dependent on the mutation status and other genetic factors of the cells treated.     

DNA-PK inhibition by NU7441 sensitizes breast cancer cells to ionizing radiation and doxorubicin

DNA-dependent protein kinase (DNA-PK) plays a key role in the repair of DNA double-strand breaks (DSBs) that are probably the most deleterious form of DNA damage. Inhibition of DNA-PK has been considered as an attractive approach to decrease resistance to therapeutically induced DNA DSBs. Ionizing radiation (IR) and doxorubicin, which induce DSBs, are used in the treatment of breast cancer. We determined the cellular concentration of DNA-PK and other DSB-activated kinases: ATM and ATR and the effect of DNA-PK inhibition by NU7441 on DNA repair, cell cycle, and survival after IR or doxorubicin treatment in three human breast cancer cell lines (MCF-7, MDA-MB-231, and T47D) representing different breast cancer subtypes. T47D cells had the highest expression of DNA-PKcs, ATM, and ATR and the most rapid rate of DNA DSB repair. IR caused a 10- to 16-fold increase in DNA-PK activity and two to threefold induction of ATM in all 3 cell lines. NU7441 inhibited IR-induced DNA-PK activity in all cell lines with IC₅₀s in the range 0.17–0.25 μM. NU7441 retarded the repair of DSB and significantly increased the sensitivity of all cell lines to IR (4- to 12-fold) and doxorubicin (3- to 13-fold). The greatest sensitiziation by NU7441 was observed in MDA-MB-231 cells. NU7441 affected the cell cycle distribution in all studied cell lines; increasing accumulation of cells in G2/M phase after DNA damage. Our data indicate that DNA-PK might be an effective target for chemo- and radio-potentiation in breast cancer and suggest that further development of DNA-PK inhibitors for clinical use is warranted.     

Preclinical evaluation of a potent novel DNA-dependent protein kinase inhibitor NU7441

DNA double-strand breaks (DSB) are the most cytotoxic lesions induced by ionizing radiation and topoisomerase II poisons, such as etoposide and doxorubicin. A major pathway for the repair of DSB is nonhomologous end joining, which requires DNA-dependent protein kinase (DNA-PK) activity. We investigated the therapeutic use of a potent, specific DNA-PK inhibitor (NU7441) in models of human cancer. We measured chemosensitization by NU7441 of topoisomerase II poisons and radiosensitization in cells deficient and proficient in DNA-PK(CS) (V3 and V3-YAC) and p53 wild type (LoVo) and p53 mutant (SW620) human colon cancer cell lines by clonogenic survival assay. Effects of NU7441 on DSB repair and cell cycle arrest were measured by gammaH2AX foci and flow cytometry. Tissue distribution of NU7441 and potentiation of etoposide activity were determined in mice bearing SW620 tumors. NU7441 increased the cytotoxicity of ionizing radiation and etoposide in SW620, LoVo, and V3-YAC cells but not in V3 cells, confirming that potentiation was due to DNA-PK inhibition. NU7441 substantially retarded the repair of ionizing radiation-induced and etoposide-induced DSB. NU7441 appreciably increased G(2)-M accumulation induced by ionizing radiation, etoposide, and doxorubicin in both SW620 and LoVo cells. In mice bearing SW620 xenografts, NU7441 concentrations in the tumor necessary for chemopotentiation in vitro were maintained for at least 4 hours at nontoxic doses. NU7441 increased etoposide-induced tumor growth delay 2-fold without exacerbating etoposide toxicity to unacceptable levels. In conclusion, NU7441 shows sufficient proof of principle through in vitro and in vivo chemosensitization and radiosensitization to justify further development of DNA-PK inhibitors for clinical use.     

Gefitinib radiosensitizes non-small cell lung cancer cells by suppressing cellular DNA repair capacity.

PURPOSE: Overexpression of the epidermal growth factor receptor (EGFR) promotes unregulated growth, inhibits apoptosis, and likely contributes to clinical radiation resistance of non-small cell lung cancer (NSCLC). Molecular blockade of EGFR signaling is an attractive therapeutic strategy for enhancing the cytotoxic effects of radiotherapy that is currently under investigation in preclinical and clinical studies. In the present study, we have investigated the mechanism by which gefitinib, a selective EGFR tyrosine kinase inhibitor, restores the radiosensitivity of NSCLC cells. EXPERIMENTAL DESIGN: Two NSCLC cell lines, A549 and H1299, were treated with 1 micromol/L gefitinib for 24 h before irradiation and then tested for clonogenic survival and capacity for repairing DNA double strand breaks (DSB). Four different repair assays were used: host cell reactivation, detection of gamma-H2AX and pNBS1 repair foci using immunofluorescence microscopy, the neutral comet assay, and pulsed-field gel electrophoresis. RESULTS: In clonogenic survival experiments, gefitinib had significant radiosensitizing effects on both cell lines. Results from all four DNA damage repair analyses in cultured A549 and H1299 cells showed that gefitinib had a strong inhibitory effect on the repair of DSBs after ionizing radiation. The presence of DSBs was especially prolonged during the first 2 h of repair compared with controls. Immunoblot analysis of selected repair proteins indicated that pNBS1 activation was prolonged by gefitinib correlating with its effect on pNBS1-labeled repair foci. CONCLUSIONS: Overall, we conclude that gefitinib enhances the radioresponse of NSCLC cells by suppressing cellular DNA repair capacity, thereby prolonging the presence of radiation-induced DSBs.     

Harmine suppresses homologous recombination repair and inhibits proliferation of hepatoma cells

To avoid cell cycle arrest or apoptosis, rapidly proliferating cancer cells have to promote DNA double strand break (DSB) repair to fix replication stress induced DSBs. Therefore, developing drugs blocking homologous recombination (HR) and nonhomologous end joining (NHEJ) – 2 major DSB repair pathways – holds great potential for cancer therapy. Over the last few decades, much attention has been paid to explore drugs targeting DSB repair pathways for cancer therapy. Here, using 2 well-established reporters for analyzing HR and NHEJ efficiency, we found that both HR and NHEJ are elevated in hepatoma cell lines Hep3B and HuH7 compared with normal liver cell lines Chang liver and QSG-7701. Our further study found that Harmine, a natural compound, negatively regulates HR but not NHEJ by interfering Rad51 recruitment, resulting in severe cytotoxicity in hepatoma cells. Furthermore, NHEJ inhibitor Nu7441 markedly sensitizes Hep3B cells to the anti-proliferative effects of Harmine. Taken together, our study suggested that Harmine holds great promise as an oncologic drug and combination of Harmine with a NHEJ inhibitor might be an effective strategy for anti-cancer treatment.     

In tumor cells regulation of DNA double strand break repair through EGF receptor involves both NHEJ and HR and is independent of p53 and K-Ras status

Purpose

The purpose of this study was to examine whether the epidermal growth factor receptor (EGFR) may be used as a general target to modulate DNA double strand break (DSB) repair in tumor cells.

Material and methods

Experiments were performed with human tumor cell lines A549, H1299 and HeLa and primate cell line CV1. EGF, ARG and TGFα were used for EGFR activation, cetuximab or erlotinib for inhibition. Overall DSB repair was assessed by γH2AX/53BP1 co-immunostaining and non-homologous end-joining (NHEJ) and homologous recombination (HR) by using NHEJ and HR reporter cells; cell cycle distribution was determined by flow cytometry and protein expression by Western blot.

Results

EGFR activation was found to stimulate overall DSB repair as well as NHEJ regardless of the ligand used. This stimulation was abolished when EGFR signaling was blocked. This regulation was found for all cell lines tested, irrespective of their p53 or K-Ras status. Stimulation and inhibition of EGFR were also found to affect HR.

Conclusions

Regulation of DSB repair by EGFR involves both the NHEJ and HR pathway, and appears to occur in most tumor cell lines regardless of p53 and K-Ras mutation status.     

Proteasome Inhibitors Diminish c-Met Expression and Induce Cell Death in Non-Small Cell Lung Cancer Cells

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and accounts for 85% of all lung carcinomas. The hepatocyte growth factor receptor (c-Met) has been considered as a potential therapeutic target for NSCLC. Proteasome inhibition induces cell apoptosis and has been used as a novel therapeutic approach for treating diseases including NSCLC; however, the effects of different proteasome inhibitors on NSCLC have not been fully investigated. The aim of this study is to determine a precise strategy for treating NSCLC by targeting c-Met using different proteasome inhibitors. Three proteasome inhibitors, bortezomib, MG132, and ONX 0914, were used in this study. Bortezomib (50 nM) significantly reduced c-Met levels and cell viability in H1299 and H441 cells, while similar effects were observed in H460 and A549 cells when a higher concentration (~100 nM) was used. Bortezomib decreased c-Met gene expression in H1299 and H441 cells, but it had no effect in A549 and H460 cells. MG-132 at a low concentration (0.5 µM) diminished c-Met levels in H441 cells, while neither a low nor a high concentration (~20 µM) altered c-Met levels in A549 and H460 cells. A higher concentration of MG-132 (5 µM) was required for decreasing c-Met levels in H1299 cells. Furthermore, MG-132 induced cell death in all four cell types. Among all the four cell lines, H441 cells expressed higher levels of c-Met and appeared to be the most susceptible to MG-132. MG-132 decreased c-Met mRNA levels in both H1299 and H441 cells. ONX 0914 reduced c-Met levels in H460, H1299, and H441 cells but not in A549 cells. c-Met levels were decreased the most in H441 cells treated with ONX 0914. ONX 0914 did not alter cell viability in H441; however, it did induce cell death among H460, A549, and H1299 cells. This study reveals that different proteasome inhibitors produce varied inhibitory effects in NSCLS cell lines.     

Adenoviral-mediated mda-7 expression suppresses DNA repair capacity and radiosensitizes non-small-cell lung cancer cells

The melanoma differentiation-associated gene-7 (mda-7) was identified by virtue of its enhanced expression in human melanoma cells induced into terminal differentiation. Enforced expression of mda-7 in human cancer cell lines of diverse origins results in the suppression of growth and induction of apoptosis. We have shown that adenoviral-mediated mda-7 (Ad-mda7) radiosensitizes non-small-cell lung cancer (NSCLC) cells by enhancing the apoptotic pathway. To identify the mechanism of this radiosensitization, we examined the level of proteins involved in the nonhomologous end-joining (NHEJ) pathway of DNA double-strand break (DSB) repair. Western blot analysis indicated that the expression of NHEJ pathway components Ku70, XRCC4, and DNA ligase IV was downregulated in NSCLC cells--A549 with Ad-mda7 treatment. No such change was observed in normal human CCD16 fibroblasts previously shown not to be radiosensitized by Ad-mda7. The biological significance of these changes of expression of proteins critical for repair of radiation-induced DSBs was confirmed via the analysis of DSB rejoining kinetics using pulsed field gel electrophoresis and assessment of host cell reactivation capacity following Ad-mda7 treatment. Based on these results, we hypothesize that Ad-mda7 sensitizes NSCLC cells to ionizing radiation by suppressing the activity of NHEJ, a pathway essential for repair of radiation-induced DSBs.     

Effect of a poly(ADP‐ribose) polymerase‐1 inhibitor against esophageal squamous cell carcinoma cell lines

Effective molecular target drugs that improve therapeutic efficacy with fewer adverse effects for esophageal cancer are highly anticipated. Poly(ADP‐ribose) polymerase (PARP) inhibitors have been proposed as low‐toxicity agents to treat double strand break (DSB)‐repair defective tumors. Several findings imply the potential relevance of DSB repair defects in the tumorigenesis of esophageal squamous cell carcinoma (ESCC). We evaluated the effect of a PARP Inhibitor (AZD2281) on the TE‐series ESCC cell lines. Of these eight cell lines, the clonogenic survival of one (TE‐6) was reduced by AZD2281 to the level of DSB repair‐defective Capan‐1 and HCC1937 cells. AZD2281‐induced DNA damage was implied by increases in γ‐H2AX and cell cycle arrest at G2/M phase. The impairment of DSB repair in TE‐6 cells was suggested by a sustained increase in γ‐H2AX levels and the tail moment calculated from a neutral comet assay after X‐ray irradiation. Because the formation of nuclear DSB repair protein foci was impaired in TE‐6 cells, whole‐exome sequencing of these cells was performed to explore the gene mutations that might be responsible. A novel mutation in RNF8, an E3 ligase targeting γ‐H2AX was identified. Consistent with this, polyubiquitination of γ‐H2AX after irradiation was impaired in TE‐6 cells. Thus, AZD2281 induced growth retardation of the DSB repair‐impaired TE‐6 cells. Interestingly, a strong correlation between basal expression levels of γ‐H2AX and sensitivity to AZD2281was observed in the TE‐series cells (R² = 0.5345). Because the assessment of basal DSB status could serve as a biomarker for selecting PARP inhibitor‐tractable tumors, further investigation is warranted.     

不同非小细胞肺癌细胞株中DNA-PKcs的表达及其与放射敏感性的关系

背景与目的：DNA依赖蛋白激酶催化亚基（DNA—dependent protein kinase catalytic subunit,DNA—PKcs）在电离辐射引起的DNA双链断裂的修复过程中起着关键作用,可影响组织细胞对放射治疗的敏感性。本研究通过检测DNA—PKcs在不同组织学类型非小细胞肺癌（non-small cell lung cancer,NSCLC）细胞中的表达情况,探讨其与放射敏感性的关系。方法：Western blot及DNA—PK活性分析法检测NSCLC细胞株A549、H1299、L78、PGCL3和H460中DNA—PKcs的含量与活性。成克隆实验分别测定各细胞株照射后的剂量一存活曲线,并分析放射敏感性与DNA—PKcs的关系。结果：成克隆实验结果显示：不同NSCLC细胞株的放射敏感性不同,A549细胞2Gy剂量照射下的存活分数（survival fraction at 2Gy,SF2）为0．74,H1299细胞为0．25,H460细胞为0．21,PGCL3细胞为0．48．L78细胞为0．58。Western blot显示各NSCLC细胞株中DNA-PKcs的表达有差异．A549细胞的DNA—PKcs表达水平为3．26±0．98,L78细胞为0．51±0．07,H1299细胞为0．51±0．11,H460细胞为0．86±0．23,PGCL3细胞为2．60±0．76。A549细胞的DNA—PKcs活性为8．30±1．03,H1299细胞为2．45±0．52,H460细胞为0．11±0．02。PGCL3细胞为4．13±0．87．L78细胞为0．42±0．07。在腺癌及大细胞癌中SF2与DNA—PKcs含量（P〈0．05,r=0．95）及活性（P=0．03,r=0．98）呈线性相关。结论：在腺癌及大细胞癌中．DNA-PKcs是判断细胞放射敏感性的重要因素。     

CD133+ cells contribute to radioresistance via altered regulation of DNA repair genes in human lung cancer cells

Background

Radioresistance in human tumors has been linked in part to a subset of cells termed cancer stem cells (CSCs). The prominin 1 (CD133) cell surface protein is proposed to be a marker enriching for CSCs. We explore the importance of DNA repair in contributing to radioresistance in CD133+ lung cancer cells.

Materials and methods

A549 and H1299 lung cancer cell lines were used. Sorted CD133+ cells were exposed to either single 4 Gy or 8 Gy doses and clonogenic survival measured. ?-H2AX immunofluorescence and quantitative real time PCR was performed on sorted CD133+ cells both in the absence of IR and after two single 4 Gy doses. Lentiviral shRNA was used to silence repair genes.

Results

A549 but not H1299 cells expand their CD133+ population after single 4 Gy exposure, and isolated A549 CD133+ cells demonstrate IR resistance. This resistance corresponded with enhanced repair of DNA double strand breaks (DSBs) and upregulated expression of DSB repair genes in A549 cells. Prior IR exposure of two single 4 Gy doses resulted in acquired DNA repair upregulation and improved repair proficiency in both A549 and H1299. Finally Exo1 and Rad51 silencing in A549 cells abrogated the CD133+ IR expansion phenotype and induced IR sensitivity in sorted CD133+ cells.

Conclusions

CD133 identifies a population of cells within specific tumor types containing altered expression of DNA repair genes that are inducible upon exposure to chemotherapy. This altered gene expression contributes to enhanced DSB resolution and the radioresistance phenotype of these cells. We also identify DNA repair genes which may serve as promising therapeutic targets to confer radiosensitivity to CSCs.     

Enhancement of Radiosensitivity by Eurycomalactone in Human NSCLC Cells Through G₂ /M Cell Cycle Arrest and Delayed DNA Double-Strand Break Repair

Radiotherapy (RT) is an important treatment for non-small cell lung cancer (NSCLC). However, the major obstacles to successful RT include the low radiosensitivity of cancer cells and the restricted radiation dose, which is given without damaging normal tissues. Therefore, the sensitizer that increases RT efficacy without dose escalation will be beneficial for NSCLC treatment. Eurycomalactone (ECL), an active quassinoid isolated from Eurycoma longifolia Jack, has been demonstrated to possess anticancer activity. In this study, we aimed to investigate the effect of ECL on sensitizing NSCLC cells to X-radiation (X-ray) as well as the underlying mechanisms. The results showed that ECL exhibited selective cytotoxicity against the NSCLC cells A549 and COR-L23 compared to the normal lung fibroblast. Clonogenic survival results indicated that ECL treatment prior to irradiation synergistically decreased the A549 and COR-L23 colony number. ECL treatment reduced the expression of cyclin B1 and CDK1/2 leading to induce cell cycle arrest at the radiosensitive G₂ /M phase. Moreover, ECL markedly delayed the repair of radiation-induced DNA double-strand breaks (DSBs). In A549 cells, pretreatment with ECL not only delayed the resolving of radiation-induced -H2AX foci but also blocked the formation of 53BP1 foci at the DSB sites. In addition, ECL pretreatment attenuated the expression of DNA repair proteins Ku-80 and KDM4D in both NSCLC cells. Consequently, these effects led to an increase in apoptosis in irradiated cells. Thus, ECL radiosensitized the NSCLC cells to X-ray via G2 /M arrest induction and delayed the repair of X-ray-induced DSBs. This study offers a great potential for ECL as an alternative safer radiosensitizer for increasing the RT efficiency against NSCLC.     

Single extreme low dose/low dose rate irradiation causes alteration in lifespan and genome instability in primary human cells

To investigate the long-term biological effect of extreme low dose ionising radiation, we irradiated normal human fibroblasts (HFLIII) with carbon ions (290 MeV u(-1), 70 keV microm(-1)) and gamma-rays at 1 mGy (total dose) once at a low dose rate (1 mGy 6-8 h(-1)), and observed the cell growth kinetics up to 5 months by continuous culturing. The growth of carbon-irradiated cells started to slow down considerably sooner than that of non-irradiated cells before reaching senescence. In contrast, cells irradiated with gamma-rays under similar conditions did not show significant deviation from the non-irradiated cells. A DNA double strand break (DSB) marker, gamma-H2AX foci, and a DSB repair marker, phosphorylated DNA-PKcs foci, increased in number when non-irradiated cells reached several passages before senescence. A single low dose/low dose rate carbon ion exposure further raised the numbers of these markers. Furthermore, the numbers of foci for these two markers were significantly reduced after the cells became fully senescent. Our results indicate that high linear energy transfer (LET) radiation (carbon ions) causes different effects than low LET radiation (gamma-rays) even at very low doses and that a single low dose of heavy ion irradiation can affect the stability of the genome many generations after irradiation.     

Silibinin inhibits human nonsmall cell lung cancer cell growth through cell‐cycle arrest by modulating expression and function of key cell‐cycle regulators

Recent studies show that silibinin possesses a strong antineoplastic potential against many cancers; however, its efficacy and underlying molecular mechanisms in nonsmall cell lung cancer (NSCLC) are not well defined. Herein, we assessed silibinin activity on prime endpoints and key molecular targets such as cell number, cell‐cycle progression, and cell‐cycle regulatory molecules in three cell lines representing different NSCLC subtypes, namely large cell carcinoma cells (H1299 and H460) and a bronchioalveolar carcinoma cell line (H322). Silibinin treatment (10–75 µM) inhibited cell growth and targeted cell‐cycle progressing causing a prominent G₁ arrest in dose‐ and time‐dependent manner. In mechanistic studies, silibinin (50–75 µM) modulated the protein levels of cyclin‐dependent kinases (CDKs) (4, 6, and 2), cyclins (D1, D3, and E), CDKIs (p18/INK4C, p21/Cip1, and p27/Kip1) in a differential manner in these three cell lines. Consistent with these observations, silibinin caused a reduction in kinase activity of CDK4 and 2 in all cell lines except no effect on CDK4 kinase activity in H460 cells, and concomitantly reduced Rb phosphorylation. Together, for the first time, these results identify potential molecular targets and anticancer effects of silibinin in NSCLC cells representing different NSCLC subtypes. © 2009 Wiley‐Liss, Inc.     

Inhibition of PI3 kinases enhances the sensitivity of non-small cell lung cancer cells to ionizing radiation

Non-small cell lung cancer (NSCLC) cells are relatively resistant to ionizing radiation (IR). The phosphatidylinositol 3 (PI3) kinases are members of a family of lipid kinases that mediate cellular functions, including cell growth, proliferation and DNA repair, which may contribute to radioresistance. We studied whether inhibition of PI3 kinases could increase the response of NSCLC cells to γ-irradiation. The results showed that pretreatment of PI3 kinase inhibitor wortmannin dose-dependently radiosensitized NSCLC A549 and H1650 cells by inhibiting colony formation, which was due to enhanced G2/M arrest and apoptosis by wortmannin. The accelerated apoptosis was accompanied by increased loss of mitochondrial membrane potential (MMP) and cytochrome c release to the cytoplasm. In addition, wortmannin pretreatment significantly increased caspase-3 activation, which was associated with the repression of X-linked inhibitor of apoptosis protein (XIAP). The radio-sensitizing effect of wortmannin was correlated with the inhibition of phosphorylated PKB/Akt level. Furthermore, wortmannin down-regulated the expression of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) which is involved in DNA double stand break (DSB) repair, as a result, leading to the inhibition of DSBs rejoining, as indicated by increased level of γ-H2AX at 24 h after IR. Taken together, our results demonstrate that wortmannin acts as a powerful radiosensitizer in NSCLC cells by inhibiting PI3K/Akt survival signaling and DNA repair protein DNA-PKcs, suggesting that PI3 kinase inhibitors may represent a novel strategy for overcoming resistance to IR-induced apoptosis in NSCLC cells.     

Roscovitine modulates DNA repair and senescence: implications for combination chemotherapy.

PURPOSE: Treatment of tumor cells by chemotherapy activates a series of responses ranging from apoptosis to premature senescence and repair. Survival responses are characterized by inhibition of cyclin-dependent kinases. Because inhibition of cyclin-dependent kinases represents a distinctive feature of DNA damage-induced prosurvival responses, we investigated the possibility that the cyclin-dependent kinase inhibitor roscovitine modulates drug-induced responses in human adenocarcinoma cells, favoring cell survival. EXPERIMENTAL DESIGN: Sublethal concentrations of doxorubicin were used to induce premature senescence in human adenocarcinoma cells. The effect of the cyclin-dependent kinase inhibitor roscovitine on the doxorubicin-dependent cell cycle checkpoint activation and DNA repair pathways was evaluated. RESULTS: Roscovitine reinforces doxorubicin-dependent G(1) checkpoint in A549 and HEC1B cells leading to decreased frequency of double-strand breaks and to the preferential induction of senescence and enhanced clonogenic survival. However, in other tumor cell lines, such as HCT116 and H1299, combined treatment with doxorubicin and roscovitine increases the frequency of double-strand breaks and dramatically sensitizes to doxorubicin. This unexpected effect of roscovitine depends on a novel ability to inhibit DNA double-strand break repair processes and requires inactivation of the pRb pathway. CONCLUSIONS: Roscovitine, by hindering DNA repair processes, has the potential to inhibit recovery of mildly damaged tumor cells after doxorubicin treatment and to increase the susceptibility of tumor cells to chemotherapy. However, in some tumor cells, the cell cycle inhibitory function of roscovitine prevails over the DNA repair inhibitory activity, favoring premature senescence and clonogenic growth. These data indicate a novel mechanism underlying combined chemotherapy, which may have wide application in treatment of carcinomas.     

Radiosensitization and DNA repair inhibition by the combined use of novel inhibitors of DNA-dependent protein kinase and poly(ADP-ribose) polymerase-1

The DNA repair enzymes, DNA-dependent protein kinase (DNA-PK) and poly(ADP-ribose) polymerase-1 (PARP-1), are key determinants of radio- and chemo-resistance. We have developed and evaluated novel specific inhibitors of DNA-PK (NU7026) and PARP-1 (AG14361) for use in anticancer therapy. PARP-1- and DNA-PK-deficient cell lines were 4-fold more sensitive to ionizing radiation (IR) alone, and showed reduced potentially lethal damage recovery (PLDR) in G(0) cells, compared with their proficient counterparts. NU7026 (10 micro M) potentiated IR cytotoxicity [potentiation factor at 90% cell kill (PF(90)) = 1.51 +/- 0.04] in exponentially growing DNA-PK proficient but not deficient cells. Similarly, AG14361 (0.4 micro M) potentiated IR in PARP-1(+/+) (PF(90) = 1.37 +/- 0.03) but not PARP-1(-/-) cells. When NU7026 and AG14361 were used in combination, their potentiating effects were additive (e.g., PF(90) = 2.81 +/- 0.19 in PARP-1(+/+) cells). Both inhibitors alone reduced PLDR approximately 3-fold in the proficient cell lines. Furthermore, the inhibitor combination completely abolished PLDR. IR-induced DNA double strand break (DNA DSB) repair was inhibited by both NU7026 and AG14361, and use of the inhibitor combination prevented 90% of DNA DSB rejoining, even 24-h postirradiation. Thus, there was a correlation between the ability of the inhibitors to prevent IR-induced DNA DSB repair and their ability to potentiate cytotoxicity. Thus, individually, or in combination, the DNA-PK and PARP-1 inhibitors act as potent radiosensitizers and show potential as tools for anticancer therapeutic intervention.     

Adenoviral-mediated PTEN expression radiosensitizes non-small cell lung cancer cells by suppressing DNA repair capacity

Expression of the PTEN tumor suppressor gene is abnormal in many human cancers. Loss of PTEN expression leads to the activation of downstream signaling pathways that have been associated with resistance to radiation. In non-small cell lung carcinoma (NSCLC), suppressed expression of PTEN is frequently due to methylation of its promoter region. In this study, we tested whether gene transfer of wild-type PTEN into an NSCLC cell line with a known methylated PTEN promoter, H1299, would increase its sensitivity to ionizing radiation. Pretreating H1299 cells with an adenoviral-mediated PTEN (Ad-PTEN)-expressing vector sensitized H1299 cells to radiation. To determine the mechanism responsible for radiosensitization, we first examined radiation-induced apoptosis, which was enhanced but did not correlate with radiosensitizing effect of Ad-PTEN. Therefore, we next examined the ability of Ad-PTEN to modulate the repair of radiation-induced DNA double-strand breaks (DSBs) using the detection of repair foci positive for gamma-H2AX, a protein that becomes evident at the sites of each DSB and that can be visualized by immunofluorescent staining. Compared with controls, the repair of radiation-induced DSBs was retarded in H1299 cells pretreated with Ad-PTEN, consistent with the radiosensitizing effect of the vector. We conclude that signal transduction pathways residing primarily in the cytoplasm may intersect with DNA damage and repair pathways in the nucleus to modulate cellular responses to radiation. Elucidating the mechanisms responsible for this intersection may lead to novel strategies for improving therapy for cancers with defective PTEN.     

Acquisition of an enhanced aggressive phenotype in human lung cancer cells selected by suboptimal doses of cisplatin following cell deattachment and reattachment

Chemotherapy is one major approach for treating non-small cell lung carcinoma (NSCLC). However, the progression-free survival rate depends on whether there is tumor metastasis after drug treatment. The biological behavior for its characteristics remains to be clarified. Here, we treated A549 and H1299 NSCLC cell lines with cisplatin, doxorubicin and gemcitabine at the IC(50) dose. Most attached cells were surviving cells (A549-A and H1299-A), whereas only a small portion of detached cells survived and reattached to tissue culture plates (A549-R and H1299-R) for further growth. Using cisplatin, a series of H1299 sublines (H1299-R2～H1299-R5) were also generated by the same selection procedure. Drug treatment increased the migratory ability of A549-R and H1299-R cells. A serial selection could enhance the invasiveness of cells. Cisplatin treatment inhibited the adhesion ability of H1299-R cells compared with their H1299 and H1299-A counterparts. H1299-R cells exhibited increased drug resistance to cisplatin and increased expression of ABCG2, CD133 and CD44. Compared with mice subcutaneously injected with H1299 cells, mice subcutaneously injected with H1299-R cells showed an increase in the number of metastatic lung nodules. We conclude that H1299-R cells selected by suboptimal doses of cisplatin following detachment from and reattachment to the tissue culture plate acquire an enhanced malignant phenotype. Therefore, they provide a more faithful lung cancer model associated with biological aggressiveness for studying clinically recurrent cancers after chemotherapy.