T-type calcium channels blockers as new tools in cancer therapies.

T-type calcium channels are involved in a multitude of cellular processes, both physiological and pathological, including cancer. T-type channels are also often aberrantly expressed in different human cancers and participate in the regulation of cell cycle progression, proliferation, migration, and survival. Here, we review the recent literature and discuss the controversies, supporting the role of T-type Ca(2+) channels in cancer cells and the proposed use of channels blockers as anticancer agents. A growing number of reports show that pharmacological inhibition or RNAi-mediated downregulation of T-type channels leads to inhibition of cancer cell proliferation and increased cancer cell death. In addition to a single agent activity, experimental results demonstrate that T-type channel blockers enhance the anticancer effects of conventional radio- and chemotherapy. At present, the detailed biological mechanism(s) underlying the anticancer activity of these channel blockers is not fully understood. Recent findings and ideas summarized here identify T-type Ca(2+) channels as a molecular target for anticancer therapy and offer new directions for the design of novel therapeutic strategies employing channels blockers. Physiological relevance: T-type calcium channels are often aberrantly expressed or deregulated in cancer cells, supporting their proliferation, survival, and resistance to treatment; therefore, T-type Ca(2+) channels could be attractive molecular targets for anticancer therapy.

PP6 regulatory subunit R1 is bidentate anchor for targeting protein phosphatase-6 to DNA-dependent protein kinase.

DNA-dependent protein kinase (DNA-PK) becomes activated in response to DNA double strand breaks, initiating repair by the non-homologous end joining pathway. DNA·PK complexes with the regulatory subunit SAPSR1 (R1) of protein phosphatase-6 (PP6). Knockdown of either R1 or PP6c prevents DNA-PK activation in response to ionizing radiation-induced DNA damage and radiosensitizes glioblastoma cells. Here, we demonstrate that R1 is necessary for and bridges the interaction between DNA-PK and PP6c. Using R1 deletion mutants, DNA-PK binding was mapped to two distinct regions of R1 spanning residues 1-326 and 522-700. Either region expressed alone was sufficient to bind DNA-PK, but only deletion of residues 1-326, not 522-700, eliminated interaction of R1 with DNA-PK. We assign 1-326 as the dominant domain and 522-700 as the supporting region. These results demonstrate that R1 acts as a bidentate anchor to DNA-PK and recruits PP6c. Targeting the dominant interface with small molecule or peptidomimetic inhibitors could specifically prevent activation of DNA-PK and thereby sensitize cells to ionizing radiation and other genotoxic agents.

The cell-cycle checkpoint kinase Chk1 is required for mammalian homologous recombination repair.

The essential checkpoint kinase Chk1 is required for cell-cycle delays after DNA damage or blocked DNA replication. However, it is unclear whether Chk1 is involved in the repair of damaged DNA. Here we establish that Chk1 is a key regulator of genome maintenance by the homologous recombination repair (HRR) system. Abrogation of Chk1 function with small interfering RNA or chemical antagonists inhibits HRR, leading to persistent unrepaired DNA double-strand breaks (DSBs) and cell death after replication inhibition with hydroxyurea or DNA-damage caused by camptothecin. After hydroxyurea treatment, the essential recombination repair protein RAD51 is recruited to DNA repair foci performing a vital role in correct HRR. We demonstrate that Chk1 interacts with RAD51, and that RAD51 is phosphorylated on Thr 309 in a Chk1-dependent manner. Consistent with a functional interplay between Chk1 and RAD51, Chk1-depleted cells failed to form RAD51 nuclear foci after exposure to hydroxyurea, and cells expressing a phosphorylation-deficient mutant RAD51(T309A) were hypersensitive to hydroxyurea. These results highlight a crucial role for the Chk1 signalling pathway in protecting cells against lethal DNA lesions through regulation of HRR.

Heavy ions, radioprotectors and genomic instability: implications for human space exploration.

The risk associated with space radiation exposure is unique from terrestrial radiation exposures due to differences in radiation quality, including linear energy transfer (LET). Both high- and low-LET radiations are capable of inducing genomic instability in mammalian cells, and this instability is thought to be a driving force underlying radiation carcinogenesis. Unfortunately, during space exploration, flight crews cannot entirely avoid radiation exposure. As a result, chemical and biological countermeasures will be an important component of successful extended missions such as the exploration of Mars. There are currently several radioprotective agents (radioprotectors) in use; however, scientists continue to search for ideal radioprotective compounds-safe to use and effective in preventing and/or reducing acute and delayed effects of irradiation. This review discusses the agents that are currently available or being evaluated for their potential as radioprotectors. Further, this review discusses some implications of radioprotection for the induction and/or propagation of genomic instability in the progeny of irradiated cells.

Deletion of the SAPS1 subunit of protein phosphatase 6 in mice increases radiosensitivity and impairs the cellular DNA damage response.

Cellular responses to DNA damage include activation of DNA-dependent protein kinase (DNA-PK) through, among others, the serine/threonine protein phosphatase 6 (PP6). We previously showed that recognition of DNA-PKcs is mediated by the SAPS1 PP6 regulatory subunit. Here, we report and characterize a SAPS1 null mouse and investigate the effects of deletion on DNA damage signaling and repair. Strikingly, neither SAPS1-null animals nor cells derived from them show gross defects, unless subjected to DNA damage by radiation or chemical agents. The overall survival of SAPS1-null animals following whole body irradiation is significantly shortened as compared to wild-type mice, and the clonogenic survival of null cells subjected to ionizing radiation is reduced. The dephosphorylation of DNA damage/repair markers, such as γH2AX, p53 and Kap1, is diminished in SAPS1-null cells as compared to wild-type controls. Our results demonstrate that loss of SAPS1 confers sensitivity to DNA damage and confirms previously reported cellular phenotypes of SAPS1 knock-down in human glioma cells. The results support a role for PP6 regulatory subunit SAPS1 in DNA damage responses, and offer a novel target for sensitization to enhance current tumor therapies, with a potential for limited deleterious side effects.

WR-1065, the active metabolite of amifostine, mitigates radiation-induced delayed genomic instability.

Compounds that can protect cells from the effects of radiation are important for clinical use, in the event of an accidental or terrorist-generated radiation event, and for astronauts traveling in space. One of the major concerns regarding the use of radio-protective agents is that they may protect cells initially, but predispose surviving cells to increased genomic instability later. In this study we used WR-1065, the active metabolite of amifostine, to determine how protection from direct effects of high- and low-LET radiation exposure influences genomic stability. When added 30 min before irradiation and in high concentrations, WR-1065 protected cells from immediate radiation-induced effects as well as from delayed genomic instability. Lower, nontoxic concentrations of WR-1065 did not protect cells from death; however, it was effective in significantly decreasing delayed genomic instability in the progeny of irradiated cells. The observed increase in manganese superoxide dismutase protein levels and activity may provide an explanation for this effect. These results confirm that WR-1065 is protective against both low- and high-LET radiation-induced genomic instability in surviving cells.

RPA mediates recombination repair during replication stress and is displaced from DNA by checkpoint signalling in human cells.

The replication protein A (RPA) is involved in most, if not all, nuclear metabolism involving single-stranded DNA. Here, we show that RPA is involved in genome maintenance at stalled replication forks by the homologous recombination repair system in humans. Depletion of the RPA protein inhibited the formation of RAD51 nuclear foci after hydroxyurea-induced replication stalling leading to persistent unrepaired DNA double-strand breaks (DSBs). We demonstrate a direct role of RPA in homology directed recombination repair. We find that RPA is dispensable for checkpoint kinase 1 (Chk1) activation and that RPA directly binds RAD52 upon replication stress, suggesting a direct role in recombination repair. In addition we show that inhibition of Chk1 with UCN-01 decreases dissociation of RPA from the chromatin and inhibits association of RAD51 and RAD52 with DNA. Altogether, our data suggest a direct role of RPA in homologous recombination in assembly of the RAD51 and RAD52 proteins. Furthermore, our data suggest that replacement of RPA with the RAD51 and RAD52 proteins is affected by checkpoint signalling.

The Relationship Between Mismatch Repair Deficiency and PD-L1 Expression in Breast Carcinoma.

Mismatch repair (MMR) deficiency in solid tumors has recently been linked to susceptibility to immunotherapies targeting the programmed cell death-1 (PD-1)/programmed cell death-1 ligand (PD-L1) axis. Loss of MMR proteins has been shown to correlate with tumoral PD-L1 expression in colorectal and endometrial carcinomas, but the association between expression of MMR proteins and PD-L1 has not previously been studied in breast carcinoma, where MMR deficiency is less common. We assessed the relationship between PD-L1 and MMR protein expression by immunohistochemistry in 245 primary and 40 metastatic breast carcinomas. Tumoral staining for PD-L1 was positive in 12% of all cases, including 32% of triple-negative cancers. MMR deficiency was observed in 0.04% of breast cancers; the single MMR-deficient case was a high-grade, triple-negative ductal carcinoma which showed dual loss of MLH1 and PMS2 proteins and expressed PD-L1. Two ER carcinomas initially were scored with MMR protein loss in tissue microarray format but were subsequently shown to be MMR-intact on whole sections. Analysis of MMR gene mutation in The Cancer Genome Atlas corroborates low frequency of MMR deficiency for invasive breast cancer. MMR protein expression is therefore unlikely to show utility as a screen for immunotherapeutic vulnerability in this tumor type, and may provoke unwarranted genetic testing in patients unlikely to have a heritable cancer syndrome. PD-L1 may be a more clinically relevant biomarker for anti-PD-1/PD-L1 therapies in this setting.

DNA crosslinking and biological activity of a hairpin polyamide-chlorambucil conjugate.

A prototype of a novel class of DNA alkylating agents, which combines the DNA crosslinking moiety chlorambucil (Chl) with a sequence-selective hairpin pyrrole-imidazole polyamide ImPy-beta-ImPy-gamma-ImPy-beta-Dp (polyamide 1), was evaluated for its ability to damage DNA and induce biological responses. Polyamide 1-Chl conjugate (1-Chl) alkylates and interstrand crosslinks DNA in cell-free systems. The alkylation occurs predominantly at 5'-AGCTGCA-3' sequence, which represents the polyamide binding site. Conjugate-induced lesions were first detected on DNA treated for 1 h with 0.1 micro M 1-Chl, indicating that the conjugate is at least 100-fold more potent than Chl. Prolonged incubation allowed for DNA damage detection even at 0.01 micro M concentration. Treatment with 1-Chl decreased DNA template activity in simian virus 40 (SV40) in vitro replication assays. 1-Chl inhibited mammalian cell growth, genomic DNA replication and cell cycle progression, and arrested cells in the G2/M phase. Moreover, cellular effects were observed at 1-Chl concentrations similar to those needed for DNA damage in cell-free systems. Neither of the parent compounds, unconjugated Chl or polyamide 1, demonstrated any cellular activity in the same concentration range. The conjugate molecule 1-Chl possesses the sequence-selectivity of a polyamide and the enhanced DNA reactivity of Chl.

T-Type Ca2+ Channel Inhibition Sensitizes Ovarian Cancer to Carboplatin.

Ovarian cancer is the deadliest gynecologic cancer, due in large part to the diagnosis of advanced stage disease, the development of platinum resistance, and inadequate treatment alternatives. Recent studies by our group and others have shown that T-type calcium (Ca(2+)) channels play a reinforcing role in cancer cell proliferation, cell-cycle progression, and apoptosis evasion. Therefore, we investigated whether T-type Ca(2+) channels affect ovarian tumor growth and response to platinum agents. Inhibition of T-type Ca(2+) channels with mibefradil or by silencing expression resulted in growth suppression in ovarian cancer cells with a simultaneous increase in apoptosis, which was accompanied by decreased expression of the antiapoptotic gene survivin (BIRC5). Analysis of intracellular signaling revealed mibefradil reduced AKT phosphorylation, increased the levels and nuclear retention of FOXO transcription factors that repress BIRC5 expression, and decreased the expression of FOXM1, which promotes BIRC5 expression. Combining carboplatin with mibefradil synergistically increased apoptosis in vitro. Importantly, mibefradil rendered platinum-resistant ovarian tumors sensitive to carboplatin in a mouse model of peritoneal metastasis. Together, the data provide rationale for future use of T-type channel antagonists together with platinum agents for the treatment of ovarian cancer.

Checkpoint Kinase 2 Negatively Regulates Androgen Sensitivity and Prostate Cancer Cell Growth.

Prostate cancer is the second leading cause of cancer death in American men, and curing metastatic disease remains a significant challenge. Nearly all patients with disseminated prostate cancer initially respond to androgen deprivation therapy (ADT), but virtually all patients will relapse and develop incurable castration-resistant prostate cancer (CRPC). A high-throughput RNAi screen to identify signaling pathways regulating prostate cancer cell growth led to our discovery that checkpoint kinase 2 (CHK2) knockdown dramatically increased prostate cancer growth and hypersensitized cells to low androgen levels. Mechanistic investigations revealed that the effects of CHK2 were dependent on the downstream signaling proteins CDC25C and CDK1. Moreover, CHK2 depletion increased androgen receptor (AR) transcriptional activity on androgen-regulated genes, substantiating the finding that CHK2 affects prostate cancer proliferation, partly, through the AR. Remarkably, we further show that CHK2 is a novel AR-repressed gene, suggestive of a negative feedback loop between CHK2 and AR. In addition, we provide evidence that CHK2 physically associates with the AR and that cell-cycle inhibition increased this association. Finally, IHC analysis of CHK2 in prostate cancer patient samples demonstrated a decrease in CHK2 expression in high-grade tumors. In conclusion, we propose that CHK2 is a negative regulator of androgen sensitivity and prostate cancer growth, and that CHK2 signaling is lost during prostate cancer progression to castration resistance. Thus, perturbing CHK2 signaling may offer a new therapeutic approach for sensitizing CRPC to ADT and radiation.

Intercalation of imidazoacridinones to DNA and its relevance to cytotoxic and antitumor activity.

Imidazoacridinones (IA) are a class of antitumor agents which includes C-1311, an interesting drug in clinical trials. This study investigated the mechanism of IA binding to DNA for a series of 13 analogs that differ in their cytotoxic potency. Using C-1311 as a model compound, crystallographic, spectroscopic and biochemical techniques were employed to characterize drug-DNA interactions. X-ray crystallographic analysis revealed a planar structure of imidazoacridinone core that is capable of intercalative DNA binding. Accordingly, C-1311 binding to DNA followed 'classical' pattern observed for intercalation, as proved by the DNA topoisomerase I-unwinding experiments, with relatively weak binding affinity (K(i)=1.2 x 10(5)M(-1)), and the binding site size of 2.4 bp. Other IA also bound to DNA with the binding affinity in the range of 10(5)M(-1) and binding site size of 2-3 bp, suggesting a prevalence of the intercalative mechanism, similar to C-1311. Considerable DNA binding affinity was displayed by all the highly cytotoxic derivatives. However, none of the analyzed drug-DNA binding parameters was significantly correlated with IA biological activities such as cell growth, DNA and RNA synthesis inhibition, or tumor growth inhibition, which suggests that the IA ability to non-covalently bind to DNA is not crucial for their biological activity. These results show that the ability to intercalate into DNA is a prominent attribute of IA, although factors other than intercalative binding seem to be required for the biological activities of IA drugs.

T-type Ca2+ channel inhibition induces p53-dependent cell growth arrest and apoptosis through activation of p38-MAPK in colon cancer cells.

UNLABELLED: Epithelial tumor cells express T-type Ca(2+) channels, which are thought to promote cell proliferation. This study investigated the cellular response to T-type Ca(2+) channel inhibition either by small-molecule antagonists or by RNAi-mediated knockdown. Selective T-type Ca(2+) channel antagonists caused growth inhibition and apoptosis more effectively in HCT116 cells expressing wild-type p53 (p53wt), than in HCT116 mutant p53(-/-) cells. These antagonists increased p53-dependent gene expression and increased genomic occupancy of p53 at specific target sequences. The knockdown of a single T-type Ca(2+) channel subunit (CACNA1G) reduced cell growth and induced caspase-3/7 activation in HCT116 p53wt cells as compared with HCT116 mutant p53(-/-) cells. Moreover, CaCo2 cells that do not express functional p53 were made more sensitive to CACNA1G knockdown when p53wt was stably expressed. Upon T-type Ca(2+) channel inhibition, p38-MAPK promoted phosphorylation at Ser392 of p53wt. Cells treated with the inhibitor SB203580 or specific RNAi targeting p38-MAPKα/ß (MAPK14/MAPK11) showed resistance to T-type Ca(2+) channel inhibition. Finally, the decreased sensitivity to channel inhibition was associated with decreased accumulation of p53 and decreased expression of p53 target genes, p21Cip1 (CDKN1A) and BCL2-binding component 3 (BBC3/PUMA). IMPLICATIONS: A novel pathway involving p53 and p38-MAPK is revealed and provides a rationale for antitumor therapies that target T-type Ca(2+) channels.

Inhibition of T-type calcium channels disrupts Akt signaling and promotes apoptosis in glioblastoma cells.

Glioblastoma multiforme (GBM) are brain tumors that are exceptionally resistant to both radio- and chemotherapy regimens and novel approaches to treatment are needed. T-type calcium channels are one type of low voltage-gated channel (LVCC) involved in embryonic cell proliferation and differentiation; however they are often over-expressed in tumors, including GBM. In this study, we found that inhibition of T-type Ca(2+) channels in GBM cells significantly reduced their survival and resistance to therapy. Moreover, either T-type selective antagonists, such as mibefradil, or siRNA-mediated knockdown of the T-type channel alpha subunits not only reduced cell viability and clonogenic potential, but also induced apoptosis. In response to channel blockade or ablation, we observed reduced phosphorylation of Akt and Rictor, suggesting inhibition of the mTORC2/Akt pathway. This was followed by reduction in phosphorylation of anti-apoptotic Bad and caspases activation. The apoptotic response was specific for T-type Ca(2+) channels, as inhibition of L-type Ca(2+) channels did not induce similar effects. Our results implicate T-type Ca(2+) channels as distinct entities for survival signaling in GBM cells and suggest that they are a novel molecular target for tumor therapy.

Inhibition of neurotensin receptor 1 selectively sensitizes prostate cancer to ionizing radiation.

Radiotherapy combined with androgen depletion is generally successful for treating locally advanced prostate cancer. However, radioresistance that contributes to recurrence remains a major therapeutic problem in many patients. In this study, we define the high-affinity neurotensin receptor 1 (NTR1) as a tractable new molecular target to radiosensitize prostate cancers. The selective NTR1 antagonist SR48692 sensitized prostate cancer cells in a dose- and time-dependent manner, increasing apoptotic cell death and decreasing clonogenic survival. The observed cancer selectivity for combinations of SR48692 and radiation reflected differential expression of NTR1, which is highly expressed in prostate cancer cells but not in normal prostate epithelial cells. Radiosensitization was not affected by androgen dependence or androgen receptor expression status. NTR1 inhibition in cancer cell-attenuated epidermal growth factor receptor activation and downstream signaling, whether induced by neurotensin or ionizing radiation, establish a molecular mechanism for sensitization. Most notably, SR48692 efficiently radiosensitized PC-3M orthotopic human tumor xenografts in mice, and significantly reduced tumor burden. Taken together, our findings offer preclinical proof of concept for targeting the NTR1 receptor as a strategy to improve efficacy and outcomes of prostate cancer treatments using radiotherapy.

Cyclophilin B expression is associated with in vitro radioresistance and clinical outcome after radiotherapy.

The tools for predicting clinical outcome after radiotherapy are not yet optimal. To improve on this, we applied the COXEN informatics approach to in vitro radiation sensitivity data of transcriptionally profiled human cells and gene expression data from untreated head and neck squamous cell carcinoma (HNSCC) and bladder tumors to generate a multigene predictive model that is independent of histologic findings and reports on tumor radiosensitivity. The predictive ability of this 41-gene model was evaluated in patients with HNSCC and was found to stratify clinical outcome after radiotherapy. In contrast, this model was not useful in stratifying similar patients not treated with radiation. This led us to hypothesize that expression of some of the 41 genes contributes to tumor radioresistance and clinical recurrence. Hence, we evaluated the expression the 41 genes as a function of in vitro radioresistance in the NCI-60 cancer cell line panel and found cyclophilin B (PPIB), a peptidylprolyl isomerase and target of cyclosporine A (CsA), had the strongest direct correlation. Functional inhibition of PPIB by small interfering RNA depletion or CsA treatment leads to radiosensitization in cancer cells and reduced cellular DNA repair. Immunohistochemical evaluation of PPIB expression in patients with HNSCC was found to be associated with outcome after radiotherapy. This work demonstrates that a novel 41-gene expression model of radiation sensitivity developed in bladder cancer cell lines and human skin fibroblasts predicts clinical outcome after radiotherapy in head and neck cancer patients and identifies PPIB as a potential target for clinical radiosensitization.

Amifostine metabolite WR-1065 disrupts homologous recombination in mammalian cells.

Repair of DNA damage through homologous recombination (HR) pathways plays a crucial role in maintaining genome stability. However, overstimulation of HR pathways in response to genotoxic stress may abnormally elevate recombination frequencies, leading to increased mutation rates and delayed genomic instability. Radiation-induced genomic instability has been detected after exposure to both low- and high-linear energy transfer (LET) radiations, but the mechanisms responsible for initiating or propagating genomic instability are not known. We have demonstrated that WR-1065, the active metabolite of amifostine, protects against radiation-induced cell killing and delayed genomic instability. We hypothesize that hyperstimulation of HR pathways plays a mechanistic role in radiation-induced genomic instability and that, in part, WR-1065 exerts it radioprotective effect through suppression of the HR pathway. Results of this study demonstrate that WR-1065 treatment selectively protected against radiation-induced cell killing in HR-proficient cell lines compared to an HR-deficient cell line. Further, WR-1065 treatment decreases HR in response to DNA damage using two different mammalian cell systems. This suppression of hyper-recombination is a previously unrecognized mechanism by which WR-1065 effects radioprotection in mammalian cells.

Lack of evidence for low-LET radiation induced bystander response in normal human fibroblasts and colon carcinoma cells.

PURPOSE: To investigate radiation-induced bystander responses and to determine the role of gap junction intercellular communication and the radiation environment in propagating this response. MATERIALS AND METHODS: We used medium transfer and targeted irradiation to examine radiation-induced bystander effects in primary human fibroblast (AG01522) and human colon carcinoma (RKO36) cells. We examined the effect of variables such as gap junction intercellular communication, linear energy transfer (LET), and the role of the radiation environment in non-targeted responses. Endpoints included clonogenic survival, micronucleus formation and foci formation at histone 2AX over doses ranging from 10-100 cGy. RESULTS: The results showed no evidence of a low-LET radiation-induced bystander response for the endpoints of clonogenic survival and induction of DNA damage. Nor did we see evidence of a high-LET, Fe ion radiation (1 GeV/n) induced bystander effect. However, direct comparison for 3.2 MeV alpha-particle exposures showed a statistically significant medium transfer bystander effect for this high-LET radiation. CONCLUSIONS: From our results, it is evident that there are many confounding factors influencing bystander responses as reported in the literature. Our observations reflect the inherent variability in biological systems and the difficulties in extrapolating from in vitro models to radiation risks in humans.

Activation of DNA-PK by ionizing radiation is mediated by protein phosphatase 6.

DNA-dependent protein kinase (DNA-PK) plays a critical role in DNA damage repair, especially in non-homologous end-joining repair of double-strand breaks such as those formed by ionizing radiation (IR) in the course of radiation therapy. Regulation of DNA-PK involves multisite phosphorylation but this is incompletely understood and little is known about protein phosphatases relative to DNA-PK. Mass spectrometry analysis revealed that DNA-PK interacts with the protein phosphatase-6 (PP6) SAPS subunit PP6R1. PP6 is a heterotrimeric enzyme that consists of a catalytic subunit, plus one of three PP6 SAPS regulatory subunits and one of three ankyrin repeat subunits. Endogenous PP6R1 co-immunoprecipitated DNA-PK, and IR enhanced the amount of complex and promoted its import into the nucleus. In addition, siRNA knockdown of either PP6R1 or PP6 significantly decreased IR activation of DNA-PK, suggesting that PP6 activates DNA-PK by association and dephosphorylation. Knockdown of other phosphatases PP5 or PP1gamma1 and subunits PP6R3 or ARS-A did not reduce IR activation of DNA-PK, demonstrating specificity for PP6R1. Finally, siRNA knockdown of PP6R1 or PP6 but not other phosphatases increased the sensitivity of glioblastoma cells to radiation-induced cell death to a level similar to DNA-PK deficient cells. Our data demonstrate that PP6 associates with and activates DNA-PK in response to ionizing radiation. Therefore, the PP6/PP6R1 phosphatase is a potential molecular target for radiation sensitization by chemical inhibition.

Cellular responses to the DNA strand-scission enediyne C-1027 can be independent of ATM, ATR, and DNA-PK kinases.

The current paradigm based upon ionizing radiation (IR) studies states that cells deficient in either ataxia-telangiectasia-mutated kinase (ATM) or related phosphatidylinositol 3 (PI 3) -kinases (ATR and DNA-PK) are hypersensitive to DNA strand breaks because they are unable to rapidly activate downstream effectors such as p53. Here we have contrasted cell responses to IR and C-1027, a radiomimetic antibiotic that induces DNA strand breaks. At equal levels of DNA double strand breaks, cell lines with inactive ATM or other phosphatidylinositol 3-kinases displayed classical hypersensitivity to IR but not to C-1027. Moreover, phosphorylation of p53 Ser-15 induced by C-1027 was independent of ATM, ATR, or DNA-PK function. We have concluded that the model based on IR studies cannot always be directly applied to DNA damage induced by other strand-scission agents.