IMT030122, A novel engineered EpCAM/CD3/4-1BB tri-specific antibody, enhances T-cell recruitment and demonstrates anti-tumor activity in mouse models of colorectal cancer.

Colorectal cancer is a major global health burden, with limited efficacy of traditional treatment modalities in improving survival rates. However, recently advances in immunotherapy has improved treatment outcomes for patients with this cancer. To address the continuing need for improved treatment efficacy, this study introduced a novel tri-specific antibody, IMT030122, that targets EpCAM, 4-1BB, and CD3. We evaluated the pharmacological efficacy and mechanism of action of IMT030122 in vitro and in vivo. In in vitro studies, IMT030122 exhibited differential binding to antigens and cells expressing EpCAM, 4-1BB, and CD3. Moreover, IMT030122 relied on EpCAM-targeted activation of intracellular CD3 and 4-1BB signaling and mediated T cell cytotoxicity specific to HCT116 colorectal cancer cells. In vivo, IMT030122 demonstrated potent anti-tumor activity, significantly inhibiting the growth of colon cancer HCT116 and MC38-hEpCAM subcutaneous grafts. Further pharmacological analysis revealed that IMT030122 recruited lymphocytes from peripheral blood into colorectal cancer tissue and exerted durable anti-tumor activity, predominantly by promoting the activation, proliferation, and differentiation of CD8T cells. Notably, IMT030122 still exhibited anti-tumor efficacy even in the presence of significantly depleted lymphocytes in colorectal cancer tissue. The potent pharmacological activity and anti-tumor effects of IMT030122 suggest it may enhance treatment efficacy and substantially extend the survival of patients with colorectal cancer in the future.

XFab-α4-1BB/CD40L fusion protein activates dendritic cells, improves expansion of antigen-specific T cells, and exhibits antitumour efficacy in multiple solid tumour models.

BACKGROUND: Additional immunotherapies are still warranted for non-responders to checkpoint inhibitors with refractory or relapsing cancers, especially for patients with "cold" tumours lacking significant immune infiltration at treatment onset. We developed XFab-α4-1BB/CD40L, a bispecific antibody targeting 4-1BB and CD40 for dendritic cell activation and priming of tumour-reactive T cells to inhibit tumours. METHODS: XFab-α4-1BB/CD40L was developed by engineering an anti-4-1BB Fab arm into a CD40L trimer based on XFab® platform. Characterisation of the bispecific antibody was performed by cell-based reporter assays, maturation of dendritic cell assays, and mixed lymphocyte reactions. The abilities of antigen-specific T-cell expansion and antitumour efficacy were assessed in syngeneic mouse tumour models. Toxicological and pharmacodynamic profiles were investigated in non-human primates. RESULTS: XFab-α4-1BB/CD40L demonstrated independent CD40 agonistic activity and conditional 4-1BB activity mediated by CD40 crosslinking, leading to dendritic cell maturation and T-cell proliferation in vitro. We confirmed the expansion of antigen-specific T cells in the vaccination model and potent tumour regression induced by the bispecific antibody alone or in combination with gemcitabine in vivo, concomitant with improved tumour-reactive T-cell infiltration. XFab-α4-1BB/CD40L showed no signs of liver toxicity at doses up to 51 mg/kg in a repeated-dose regimen in non-human primates. CONCLUSIONS: XFab-α4-1BB/CD40L is capable of enhancing antitumour immunity by modulating dendritic cell and T-cell functions via targeting 4-1BB agonism to areas of CD40 expression. The focused, potent, and safe immune response induced by the bispecific antibody supports further clinical investigations for the treatment of solid tumours.

Anti-c-MET Fab-Grb2-Gab1 Fusion Protein-Mediated Interference of c-MET Signaling Pathway Induces Methuosis in Tumor Cells.

Bio-macromolecules have potential applications in cancer treatment due to their high selectivity and efficiency in hitting therapeutic targets. However, poor cell membrane permeability has limited their broad-spectrum application in cancer treatment. The current study developed highly internalizable anti-c-MET antibody Fab fusion proteins with intracellular epitope peptide chimera to achieve the dual intervention from the extracellular to intracellular targets in tumor therapy. In vitro experiments demonstrated that the fusion proteins could interfere with the disease-associated intracellular signaling pathways and inhibit the uncontrolled proliferation of tumor cells. Importantly, investigation of the underlying mechanism revealed that these protein chimeras could induce vacuolation in treated cells, thus interfering with the normal extension and arrangement of microtubules as well as the mitosis, leading to the induction of methuosis-mediated cell death. Furthermore, in vivo tumor models indicated that certain doses of fusion proteins could inhibit the A549 xenograft tumors in NOD SCID mice. This study thus provides new ideas for the intracellular delivery of bio-macromolecules and the dual intervention against tumor cell signaling pathways.

Preclinical characterization of a Fab-like CD3/CLDN18.2 XFab® bispecific antibody against solid tumors.

The claudin 18.2(CLDN18.2) antigen is highly expressed in gastric mucosa epithelial cells and frequently expressed in malignant tumors. Positive clinical outcomes have popularized claudin 18.2 as a novel cellular and antibody therapeutic. Here, we designed a bispecific antibody-ZWB67 using the XFab® platform, aimed at redirecting CD3+ effector T cells to CLDN18.2+ target cells or tissues. Physicochemical characterization, binding properties, T cell stimulatory activity, and T cell-dependent cellular cytotoxicity of ZWB67 were evaluated in dosage intervals using antigens of CD3 and target cells expressing CLDN18.2 or CD3. Then, the anti-tumor activity was assessed in humanized CD3EDG mice bearing MC-38-hCLDN18.2 tumors. Our data demonstrate that ZWB67 specifically binds to the human CD3e antigen (KD = 1.04E-08 M) and binds more strongly to CLDN18.2+ cells than to CD3+ cells (4.3- to 9.2-fold difference). ZWB67 showed good activity in the luciferase reporter system and exhibited dose-dependent activation, cytotoxicity of T cells, and cytokine release when co-cultured with CLDN18.2+ cells and CD3+ T cells. ZWB67 also exhibited high in vivo efficacy in the MC-38-hCLDN18.2 xenograft mouse model. In conclusion, the novel anti-CLDN18.2 × anti-CD3 bispecific antibody exhibited low affinity for anti-CD3, highly specific binding, potent cytotoxicity, and anti-tumor activity. These data provide a basis for future preclinical and clinical development of this therapeutic strategy.

Methods for PTEN in Stem Cells and Cancer Stem Cells.

PTEN (phosphatase and tensin homologue) is the first tumor suppressor identified to have phosphatase activity and its gene is the second most frequently deleted or mutated tumor-suppressor gene associated with human cancers. Germline PTEN mutations are the cause of three inherited autosomal dominant disorders. Phosphatidylinositol 3,4,5,-triphosphate (PIP3), the product of the PI3 kinase, is one of the key intracellular targets of PTEN's phosphatase activity, although PTEN's phosphatase-independent activities have also been identified. PTEN is critical for stem cell maintenance, which contributes to its controlled tumorigenesis. PTEN loss leads the development of cancer stem cells (CSCs) that share properties with somatic stem cells, including the capacity for self-renewal and multi-lineage differentiation. Methods to isolate and functionally test stem cells and CSCs are important for understanding PTEN functions and the development of therapeutic approaches to target CSCs without having adverse effects on normal stem cells. Here, we describe protocols for the isolation and functional analysis of PTEN deficient embryonic stem cells, hematopoietic stem cells and leukemia-initiating cells (LICs), neural stem cells, and prostate stem cells and CSCs.

TNKS1BP1 functions in DNA double-strand break repair though facilitating DNA-PKcs autophosphorylation dependent on PARP-1.

TNKS1BP1 was originally identified as an interaction protein of tankyrase 1, which belongs to the poly(ADP-ribose) polymerase (PARP) superfamily. PARP members play important roles for example in DNA repair, telomere stability and mitosis regulation. Although the TNKS1BP1 protein was considered to be a poly(ADP-ribosyl)ation acceptor of tankyrase 1, its function is still unknown. Here we firstly identified that TNKS1BP1 was up-regulated by ionizing radiation (IR) and the depletion of TNKS1BP1 significantly sensitized cancer cells to IR. Neutral comet assay, pulsed-field gel electrophoresis, and γH2AX foci analysis indicated that TNKS1BP1 is required for the efficient repair of DNA double-strand breaks (DSB). The TNKS1BP1 protein was demonstrated to interact with DNA-dependent protein kinase (DNA-PKcs) and poly(ADP-ribose) polymerase 1 (PARP-1), by co-immunoprecipitation analysis. Moreover, TNKS1BP1 was shown to promote the association of PARP-1 and DNA-PKcs. Overexpression of TNKS1BP1 induced the autophosphorylation of DNA-PKcs/Ser2056 in a PARP-1 dependent manner, which contributed to an increased capability of DNA DSB repair. Inhibition of PARP-1 blocked the TNKS1BP1-mediated DNA-PKcs autophosphorylation and attenuated the PARylation of DNA-PKcs. TNKS1BP1 is a newly described component of the DNA DSB repair machinery, which provides much more mechanistic evidence for the rationale of developing effective anticancer measures by targeting PARP-1 and DNA-PKcs.

PIG3 functions in DNA damage response through regulating DNA-PKcs homeostasis.

The p53-inducible gene 3 (PIG3) recently has been reported to be a new player in DNA damage signaling and response, but the crucial mechanism remains unclear. In the present study, the potential mechanism of PIG3 participation in the DNA damage response induced by ionizing radiation (IR) was investigated in multiple cell lines with depleted expression of PIG3 transiently or stably by the small interference RNA and lentivirus-mediated shRNA expression strategies. PIG3 knockdown led to an abnormal DNA damage response, including decreased IR-induced phosphorylation of H2AX, Chk1, Chk2 and Kap-1 as well as a prolonged G2-M arrest and aberrant mitotic progression. Notably, PIG3 knockdown resulted in a striking depression of cellular DNA-PKcs protein level, and was accompanied by a downregulation of ATM. Re-expression of PIG3 effectively rescued the depression of DNA-PKcs in PIG3-depleted cells. This negative regulation of DNA-PKcs by depleting PIG3 seemed to take place at the translational level but not at the levels of transcription or protein degradation. However, a compensatory feedback of increased mRNA expression of DNA-PKcs was formed in PIG3-depleted cells after a few passages or cell cycles of subculture, which led the recovery of the DNA-PKcs protein level and the consequent recovered efficiency of the DNA damage response. These results provide a new insight into the mechanism of PIG3's functioning in DNA damage signaling and the regulation network of cellular DNA-PKcs expression homeostasis.

Proteomic profiling revealed the functional networks associated with mitotic catastrophe of HepG2 hepatoma cells induced by 6-bromine-5-hydroxy-4-methoxybenzaldehyde.

Mitotic catastrophe, a form of cell death resulting from abnormal mitosis, is a cytotoxic death pathway as well as an appealing mechanistic strategy for the development of anti-cancer drugs. In this study, 6-bromine-5-hydroxy-4-methoxybenzaldehyde was demonstrated to induce DNA double-strand break, multipolar spindles, sustain mitotic arrest and generate multinucleated cells, all of which indicate mitotic catastrophe, in human hepatoma HepG2 cells. We used proteomic profiling to identify the differentially expressed proteins underlying mitotic catastrophe. A total of 137 differentially expressed proteins (76 upregulated and 61 downregulated proteins) were identified. Some of the changed proteins have previously been associated with mitotic catastrophe, such as DNA-PKcs, FoxM1, RCC1, cyclin E, PLK1-pT210, 14-3-3σ and HSP70. Multiple isoforms of 14-3-3, heat-shock proteins and tubulin were upregulated. Analysis of functional significance revealed that the 14-3-3-mediated signaling network was the most significantly enriched for the differentially expressed proteins. The modulated proteins were found to be involved in macromolecule complex assembly, cell death, cell cycle, chromatin remodeling and DNA repair, tubulin and cytoskeletal organization. These findings revealed the overall molecular events and functional signaling networks associated with spindle disruption and mitotic catastrophe.

Structure of poly(ethylene glycol)-water mixture studied by polymer reference interaction site model theory.

In this work, the polymer reference interaction site model is applied to investigate the structure of poly(ethylene glycol) (PEG) aqueous solution with the strong hydrogen-bond interactions. In the theoretical model, the renormalized technique of electrostatic potentials is combined with our recently proposed multisite semiflexible chain model to describe the inter- and intramolecular correlations. To test the model for the description of hydrogen bonding, the intermolecular correlation functions of water, ethylene glycol (EG), and EG-water binary mixture are calculated. The results are in good agreement with the corresponding simulation or experimental data. The validated model is then employed to predict the intermolecular correlation functions of different sites of the PEG and its aqueous solution. Another priority of the model is that it can obtain the corresponding direct correlation functions directly.

Radiosensitization and growth inhibition of cancer cells mediated by an scFv antibody gene against DNA-PKcs in vitro and in vivo.

BACKGROUND: Overexpression of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is commonly occurred in cancers and causes radioresistance and poor prognosis. In present study, the single-chain variable antibody fragments (scFv) targeting DNA-PKcs was developed for the application of radiosensitization in vitro and in vivo. A humanized semisynthetic scFv library and the phage-display antibodies technology were employed to screen DNA-PKcs scFv antibody. METHODS: DNA-PKcs epitopes were predicted and cloned. A humanized semisynthetic scFv library and the phage-display antibodies technology were employed to screen DNA-PKcs scFv antibody. DNA damage repair was analyzed by comet assay and immunofluorescence detection of gammaH2AX foci. The radiosensitization in vivo was determined on Balb/c athymic mice transplanted tumours of HeLa cells. RESULTS: Four epitopes of DNA-PKcs have been predicted and expressed as the antigens, and a specific human anti-DNA-PKcs scFv antibody gene, anti-DPK3-scFv, was obtained by screening the phage antibody library using the DNA-PKcs peptide DPK3. The specificity of anti-DPK3-scFv was verified, in vitro. Transfection of HeLa cells with the anti-DPK3-scFv gene resulted in an increased sensitivity to IR, decreased repair capability of DNA double-strand breaks (DSB) detected by comet assay and immunofluorescence detection of gammaH2AX foci. Moreover, the kinase activity of DNA-PKcs was inhibited by anti-DPK3-scFv, which was displayed by the decreased phosphorylation levels of its target Akt/S473 and the autophosphorylation of DNA-PKcs on S2056 induced by radiation. Measurement of the growth and apoptosis rates showed that anti-DPK3-scFv enhanced the sensitivity of tumours transplanted in Balb/c athymic mice to radiation therapy. CONCLUSION: The antiproliferation and radiosensitizing effects of anti-DPK3-scFv via targeting DNA-PKcs make it very appealing for the development as a novel biological radiosensitizer for cancer therapeutic potential.

Inactivation of DNA-dependent protein kinase leads to spindle disruption and mitotic catastrophe with attenuated checkpoint protein 2 Phosphorylation in response to DNA damage.

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is well known as a critical component involving the nonhomologous end joining pathway of DNA double-strand breaks repair. Here, we showed another important role of DNA-PKcs in stabilizing spindle formation and preventing mitotic catastrophe in response to DNA damage. Inactivation of DNA-PKcs by small interfering RNA or specific inhibitor NU7026 resulted in an increased outcome of polyploidy after 2-Gy or 4-Gy irradiation. Simultaneously, a high incidence of multinucleated cells and multipolar spindles was detected in DNA-PKcs-deficient cells. Time-lapse video microscopy revealed that depression of DNA-PKcs results in mitotic catastrophe associated with mitotic progression failure in response to DNA damage. Moreover, DNA-PKcs inhibition led to a prolonged G(2)-M arrest and increased the outcome of aberrant spindles and mitotic catastrophe in Ataxia-telangiectasia mutated kinase (ATM)-deficient AT5BIVA cells. We have also revealed the localizations of phosphorylated DNA-PKcs/T2609 at the centrosomes, kinetochores, and midbody during mitosis. We have found that the association of DNA-PKcs and checkpoint kinase 2 (Chk2) is driven by Ku70/80 heterodimer. Inactivation of DNA-PKcs strikingly attenuated the ionizing radiation-induced phosphorylation of Chk2/T68 in both ATM-efficient and ATM-deficient cells. Chk2/p-T68 was also shown to localize at the centrosomes and midbody. These results reveal an important role of DNA-PKcs on stabilizing spindle formation and preventing mitotic catastrophe in response to DNA damage and provide another prospect for understanding the mechanism coupling DNA repair and the regulation of mitotic progression.

HIV-1 Tat regulates cyclin B1 by promoting both expression and degradation.

Cyclin B1, an important cell cycle regulator, was up-regulated in lymphocytes of human immunodeficiency virus (HIV)-infected patients. However, the mechanism of cyclin B1 up-regulation and the effects of the up-regulation on the host cells remain unclear. Here, we show that HIV-encoded Tat protein regulates cyclin B1 levels in two different ways: first, Tat stimulates the transcription of cyclin B1, which increases cyclin B1 levels and promotes the cells apoptosis; and second, Tat stimulates polyubiquitination-mediated degradation of cyclin B1 through binding to the N-terminal of cyclin B1 (aa 61-129) that is just downstream of the D box, which prevents excessive levels of cyclin B1 in the cells. These results suggest that Tat-regulating cyclin B1 affects the status of HIV: Tat stimulates cyclin B1 expression to slow down the host cell cycle progress and to promote the host cell apoptosis, which might facilitate HIV release; Tat stimulates cyclin B1 degradation to prevent overaccumulation of cyclin B1, which might facilitate HIV replication. Taken together, our results reveal for the first time how HIV-Tat regulates cyclin B1 and keeps its balance in the cells.

[Combination with SN-38 on human colon cancer LoVo cells].

OBJECTIVE: To observe the anti-proliferation effect of bevacizumab and SN-38 (active metabolite of irinotecan), and investigate the possible mechanisms of these two agents. METHODS: Human colon cancer LoVo cells were cultured under hypoxic conditions. Inhibition of cell proliferation was evaluated by MTT assay. The drug modulation on HIF-1alpha, VEGF, ERK and AKT were assessed by the following assays. The mRNA expression of HIF-1alpha and VEGF were measured by RT-PCR. The protein expression of HIF-1alpha, ERK and AKT were evaluated by Western blot analysis, and VEGF by ELISA assay. RESULTS: Among different combination schedules, Bevacizumab given after SN-38 show most synergistic anti-proliferation effect. Under hypoxic conditions, the expression of HIF-1alpha and VEGF increased as time accumulated, Bevacizumab combined with SN-38 almost completely inhibited the expression of HIF-1alpha and VEGF. Moreover, the MAP kinase pathway was involved in the drug modulation of HIF-1alpha and VEGF. CONCLUSION: These findings suggest the anti-proliferation effect of bevacizumab and SN-38 was schedule-dependent, and the synergistic effect of Bevacizumab and SN-38 was related to drug modulation of the HIF-1alpha and MAP kinase pathway.

DNA-dependent protein kinase catalytic subunit modulates the stability of c-Myc oncoprotein.

BACKGROUND: C-Myc is a short-lived oncoprotein that is destroyed by ubiquitin-mediated proteolysis. Dysregulated accumulation of c-Myc commonly occurs in human cancers. Some of those cases with the dysregulated c-Myc protein accumulation are attributed to gene amplification or increased mRNA expression. However, the abnormal accumulation of c-Myc protein is also a common finding in human cancers with normal copy number and transcription level of c-Myc gene. It seems that the mechanistic dysregulation in the control of c-Myc protein stabilization is another important hallmark associated with c-Myc accumulation in cancer cells. Here we report a novel mechanistic pathway through which DNA-dependent protein kinase catalytic subunit (DNA-PKcs) modulates the stability of c-Myc protein. RESULTS: Firstly, siRNA-mediated silencing of DNA-PKcs strikingly downregulated c-Myc protein levels in HeLa and HepG2 cells, and simultaneously decreased cell proliferation. The c-Myc protein level in DNA-PKcs deficient human glioma M059J cells was also found much lower than that in DNA-PKcs efficient M059K cells. ATM deficiency does not affect c-Myc expression level. Silencing of DNA-PKcs in HeLa cells resulted in a decreased stability of c-Myc protein, which was associated the increasing of c-Myc phosphorylation on Thr58/Ser62 and ubiquitination level. Phosphorylation of Akt on Ser473, a substrate of DNA-PKcs was found decreased in DNA-PKcs deficient cells. As the consequence, the phosphorylation of GSK3 beta on Ser9, a negatively regulated target of Akt, was also decreased, and which led to activation of GSK 3beta and in turn phosphorylation of c-Myc on Thr58. Moreover, inhibition of GSK3 activity by LiCl or specific siRNA molecules rescued the downregulation of c-Myc mediated by silencing DNA-PKcs. Consistent with this depressed DNA-PKcs cell model, overexpressing DNA-PKcs in normal human liver L02 cells, by sub-chronically exposing to very low dose of carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), increased c-Myc protein level, the phosphorylation of Akt and GSK3 beta, as well as cell proliferation. siRNA-mediated silencing of DNA-PKcs in this cell model reversed above alterations to the original levels of L02 cells. CONCLUSION: A suitable DNA-PKcs level in cells is necessary for maintaining genomic stability, while abnormal overexpression of DNA-PKcs may contribute to cell proliferation and even oncogenic transformation by stabilizing the c-Myc oncoprotein via at least the Akt/GSK3 pathway. Our results suggest DNA-PKcs a novel biological role beyond its DNA repair function.

Radioprotection of 4-hydroxy-3,5-dimethoxybenzaldehyde (VND3207) in culture cells is associated with minimizing DNA damage and activating Akt.

Vanillin is a naturally occurring compound and food-flavoring agent with antioxidant and antimutagenic activities. In present study, we explored the radioprotective effect of a novel vanillin derivative VND3207 (4-hydroxy-3,5-dimethoxybenzaldehyde). VND3207 has a much higher potential in scavenging hydroxyl radical and superoxide radical than vanillin as indicated in the ESR spin-trapping measurement, and it can effectively protect plasmid DNA against 10-50 Gy gamma-ray induced breaks in vitro at the concentrations as low as 10-20 microM. Using human lymphoblastoid AHH-1 cells and human fibroblastoid HFS cells, we demonstrated that VND3207 at 5-40 microM concentrations significantly attenuated the inhibition of proliferation and occurrence of apoptosis produced by 1-8 Gy gamma-irradiation. In the cultured cells, VND3207 significantly decreased the initial production and residual level of DNA double-strand breaks (DSBs) induced by 2 or 8 Gy irradiation. Treatment of VND3207 enhanced the level of DNA-PKcs protein, a critical component of DNA DSB repair pathway in the cells with or without gamma-irradiation. Consistently, the phosphorylation of Akt protein, a mediator of survival signal, as well as its substrate GSK3beta was concurrently increased by VND3207. Our results suggest that VND3207 has radioprotection effect through its capabilities as a powerful antioxidant, in minimizing DNA damage, and activating survival signal Akt pathway, and it may be of value in the development of radioprotective compounds.

[Effect of small interfering RNA-induced LKB1 gene silencing on the biological behavior of lung carcinoma cells].

OBJECTIVE: To study the effect of the small interfering RNA (siRNA)-mediated LKB1 gene silencing on the biological behavior of lung carcinoma cells. METHODS: RNA interference technique was used to silence LKB1 gene in lung carcinoma cells, and the expression of LKB1 protein were subsequently detected by Western blotting. The cell proliferation was then assessed by observing the growth curve and clone formation of the cells, and cell cycle and apoptosis were assayed by flow cytometry. RESULTS: LKB1 siRNA resulted in remarkable suppression of LKB1 expression in the lung carcinoma cells. LKB1 gene silencing resulted in accelerated cell proliferation, but cell apoptosis underwent no significant changes. CONCLUSION: Down-regulation of LKB1 gene expression can stimulate malignant biological behavior of lung carcinoma cells.

Dose-dependent expression changes of early response genes to ionizing radiation in human lymphoblastoid cells.

The sensitivity of cancer cells as well as normal cells in response to ionizing radiation (IR) is believed to be associated with the early inducible expression of specific genes. Using cDNA microarray technology, here we explored and compared the global transcriptional changes in human lymphoblastoid AHH-1 cells irradiated with 0.05-, 0.2-, 0.5-, 2.0- and 10-Gy doses of gamma-rays 4 h after exposure. A dose as low as 0.05 Gy was efficient in inducing a transcriptional response including the up-regulation of 25 genes, some of which are involved in signal transduction pathways, e.g. BMPR2, GPR124, MAPK8IP2 and AGGF1, and the down-regulation of 18 genes. Expression of some genes was altered only at a specific dose. Most importantly, we discovered a number of radiation-response genes, e.g. DNA repair gene XPC, tumor protein p53 inducible protein 3 gene (TP53I3), immediate early response 5 gene, whose transcriptional levels were increased or depressed by IR in a dose-dependent trend within the dose range 0.05-10 Gy. The dose-dependent induced expression of TP53I3 and XPC was confirmed by Northern blot analyses. Using quantitative real-time PCR, we further confirmed that XPC gene induction was dose dependent as well as time dependent, reaching a peak 4 h post-2 Gy and 10 h post-0.05 Gy. The maximum induced expression level of the XPC gene was higher after 2 Gy (3.2-fold) than 0.05 Gy (1.93-fold). The identification of these radiation-inducible genes, especially those exhibiting a dose-dependent response, not only expands our knowledge of the mechanisms underlying the diverse biological effects induced by IR, but provides candidates for developing novel biomarkers of radiation injury.

Vanillin derivative 6-bromine-5-hydroxy-4-methoxybenzaldehyde-elicited apoptosis and G2/M arrest of Jurkat cells proceeds concurrently with DNA-PKcs cleavage and Akt inactivation.

Vanillin, a naturally occurring food component, has been reported to have anti-mutagenic and anti-metastatic potentials, and to inhibit DNA-PKcs activity. However, vanillin itself exhibits very weak antiproliferative activity. We explored the effects of bromovanin (6-bromine-5-hydroxy-4-methoxybenzaldehyde), a novel vanillin derivative, on survival and cell-cycle progression of human Jurkat leukemia cells. Treatment with >10 microM bromovanin significantly elicited apoptosis and G2/M arrest in Jurkat cells in a dose- and time-dependent manner. Bromovanin-induced DNA double-strand breaks (DSB) were demonstrated by means of comet assay as well as detection of phosphorylated H2AX, a sensitive indicator of DNA DSBs. Immuno-hybridization analysis revealed that the cleavage of procaspase-3 and DNA-PKcs occurred concurrently with bromovanin-induced apoptosis. Furthermore, phosphorylated Akt protein (Ser473), which is catalyzed by DNA-PKcs, as well as phosphorylated GSK3beta (a substrate of activated Akt), markedly decreased in bromovanin-treated Jurkat cells, suggesting that bromovanin leads to inactivation of Akt pathway via cleaving DNA-PKcs. These multiple effects, associated with the regimen of cancer therapeutic strategies, make bromovanin very appealing for future development as a novel anticancer drug.

HIV-1 Tat depresses DNA-PK(CS) expression and DNA repair, and sensitizes cells to ionizing radiation.

PURPOSE: There is accumulating evidence that cancer patients with human immmunodeficiency virus-1/acquired immunodeficency syndrome (HIV-1/AIDS) have more severe tissue reactions and often develop cutaneous toxic effects when subjected to radiotherapy. Here we explored the effects of the HIV-1 Tat protein on cellular responses to ionizing radiation. METHODS AND MATERIALS: Two Tat-expressing cell lines, TT2 and TE671-Tat, were derived from human rhabdomyosarcoma cells by transfecting with the HIV-1 tat gene. Radiosensitivity was determined using colony-forming ability. Gene expression was assessed by cDNA microarray and immunohybridization. The Comet assay and gamma-H2AX foci were use to detect DNA double-strand breaks (DSBs) and repair. Radiation-induced cell cycle changes were detected by flow cytometry. RESULTS: The radiosensitivity of TT2 and TE671-Tat cells was significantly increased as compared with parental TE671 cells or the control TE671-pCI cells. Tat also increased proliferation activity. The comet assay and gammaH2AX foci detection revealed a decreased capacity to repair radiation-induced DNA DSBs in Tat-expressing cells. Microarray assay demonstrated that the DNA repair gene DNA-PKcs, and cell cycle-related genes Cdc20, Cdc25C, KIF2C and CTS1 were downregulated in Tat-expressing cells. Depression of DNA-PKcs in Tat-expressing cells was further confirmed by RT-PCR and immuno-hybridization analysis. Tat-expressing cells exhibited a prolonged S phase arrest after 4 Gy gamma-irradiation, and a noticeable delay in the initiation and elimination of radiation-induced G(2)/M arrest as compared with parental cells. In addition, the G(2)/M arrest was incomplete in TT2 cells. Moreover, HIV-1 Tat resulted in a constitutive overexpression of cyclin B1 protein. CONCLUSION: HIV-1 Tat protein sensitizes cells to ionizing radiation via depressing DNA repair and dysregulating cell cycle checkpoints. These observations provide new insight into the increased tissue reactions of AIDS cancer patients to radiotherapy.

Silencing of DNA-PKcs alters the transcriptional profile of certain signal transduction genes related to proliferation and differentiation in HeLa cells.

DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a member of a sub-family of phosphoinositol 3-kinases, has been reported overexpressed in various human cancers, but its significance is unclear. In the present study, we generated the stable cell line HeLa(siRNAH1) of silenced DNA-PKcs by transfecting HeLa cells with the siRNA construct targeting the catalytic motif of DNA-PKcs. The expression of DNA-PKcs was markedly suppressed in HeLa(siRNAH1) cells, and eventuating in increased cellular sensitivity to ionizing radiation as well as cisplatin. Microarray assay was used to explore the transcriptional profiling of signal transduction-associated genes. The results demonstrated that 15 genes were up-regulated and eight were down-regulated in HeLa(siRNAH1) as compared with the HeLa(control) cells that transfected with non-specific siRNA construct. Seven of the up-regulated genes are associated with the interferon-signaling events, the others function in the BMP signal pathway, or as regulators of cell cycle and differentiation. The down-regulated genes include IL8, IL10RA, DAPK3, and those involved in nuclear factor of activated T cells (NFAT) signal pathway and endocrine responsiveness. Using the NFAT-driving secreted alkaline phosphatase reporter expression system, we further confirmed that NFAT transcriptional activity was markedly minimized after silencing DNA-PKcs. These results demonstrated that inactivation of DNA-PKcs altered the transcriptional level of certain signal transduction-associated genes related to proliferation and differentiation.