Preclinical studies of cranial radiation therapy (RT) using animal brain tumor models have been hampered by technical limitations in the delivery of clinically relevant RT. We established a bioimageable mouse model of glioblastoma multiforme (GBM) and an image-guided radiation delivery system that facilitated precise tumor localization and treatment and which closely resembled clinical RT. Our novel radiation system makes use of magnetic resonance imaging (MRI) and bioluminescent imaging (BLI) to define tumor volumes, computed tomographic (CT) imaging for accurate treatment planning, a novel mouse immobilization system, and precise treatments delivered with the Small Animal Radiation Research Platform. We demonstrated that, in vivo, BLI correlated well with MRI for defining tumor volumes. Our novel restraint system enhanced setup reproducibility and precision, was atraumatic, and minimized artifacts on CT imaging used for treatment planning. We confirmed precise radiation delivery through immunofluorescent analysis of the phosphorylation of histone H2AX in irradiated brains and brain tumors. Assays with an intravenous near-infrared fluorescent probe confirmed that radiation of orthografts increased disruption of the tumor blood-brain barrier (BBB). This integrated model system, which facilitated delivery of precise, reproducible, stereotactic cranial RT in mice and confirmed RT's resultant histologic and BBB changes, may aid future brain tumor research.

Histone deacetylases mediate critical cellular functions but relatively little is known about mechanisms controlling their expression, including expression of HDAC4, a class II HDAC implicated in the modulation of cellular differentiation and viability. Endogenous HDAC4 mRNA, protein levels and promoter activity were all readily repressed by mithramycin, suggesting regulation by GC-rich DNA sequences. We validated consensus binding sites for Sp1/Sp3 transcription factors in the HDAC4 promoter through truncation studies and targeted mutagenesis. Specific and functional binding by Sp1/Sp3 at these sites was confirmed with chromatin immunoprecipitation (ChIP) and electromobility shift assays (EMSA). Cotransfection of either Sp1 or Sp3 with a reporter driven by the HDAC4 promoter led to high activities in SL2 insect cells (which lack endogenous Sp1/Sp3). In human cells, restored expression of Sp1 and Sp3 up-regulated HDAC4 protein levels, whereas levels were decreased by RNA-interference-mediated knockdown of either protein. Finally, variable levels of Sp1 were in concordance with that of HDAC4 in a number of human tissues and cancer cell lines. These studies together characterize for the first time the activity of the HDAC4 promoter, through which Sp1 and Sp3 modulates expression of HDAC4 and which may contribute to tissue or cell-line-specific expression of HDAC4.

Anumber of proteins are recruited to nuclear foci upon exposure to double-strand DNA damage, including 53BP1 and Rad51, but the precise role of these DNA damage–induced foci remain unclear. Here we show in a variety of human cell lines that histone deacetylase (HDAC) 4 is recruited to foci with kinetics similar to, and colocalizes with, 53BP1 after exposure to agents causing double-stranded DNA breaks. HDAC4 foci gradually disappeared in repair-proficient cells but persisted in repair-deficient cell lines or cells irradiated with a lethal dose, suggesting that resolution of HDAC4 foci is linked to repair. Silencing of HDAC4 via RNA interference surprisingly also decreased levels of 53BP1 protein, abrogated the DNA damage–induced G2 delay, and radiosensitized HeLa cells. Our combined results suggest that HDAC4 is a critical component of the DNA damage response pathway that acts through 53BP1 and perhaps contributes in maintaining the G2 cell cycle checkpoint.

HIV-1 integration is mediated by the HIV-1 integrase protein, which joins 3'-ends of viral DNA to host cell DNA. To complete the integration process, HIV-1 DNA has to be joined to host cell DNA also at the 5'-ends. This process is called post-integration repair (PIR). Integration and PIR involve a number of cellular co-factors. These proteins exhibit different degrees of involvement in integration and/or PIR. Some are required for efficient integration or PIR. On the other hand, some reduce the efficiency of integration. Finally, some are involved in integration site selection. We have studied the role of the histone deacetylase HDAC4 in these processes. HDAC4 was demonstrated to play a role in both cellular double-strand DNA break repair and transcriptional regulation. We observed that HDAC4 associates with viral DNA in an integrase-dependent manner. Moreover, infection with HIV-1-based vectors induces foci of the HDAC4 protein. The related histone deacetylases, HDAC2 and HDAC6, failed to associate with viral DNA after infection. These data suggest that HDAC4 accumulates at integration sites. Finally, overexpression studies with HDAC4 mutants suggest that HDAC4 may be required for efficient transduction by HIV-1-based vectors in cells that are deficient in other DNA repair proteins. We conclude that HDAC4 is likely involved in PIR.

The class II Histone deacetylase (HDAC), HDAC4, is expressed in a tissue-specific manner, and it represses differentiation of specific cell types. We demonstrate here that HDAC4 is expressed in the proliferative zone in small intestine and colon and that its expression is down-regulated during intestinal differentiation in vivo and in vitro. Subcellular localization studies demonstrated HDAC4 expression was predominantly nuclear in proliferating HCT116 cells and relocalized to the cytoplasm after cell cycle arrest. Down-regulating HDAC4 expression by small interfering RNA (siRNA) in HCT116 cells induced growth inhibition and apoptosis in vitro, reduced xenograft tumor growth, and increased p21 transcription. Conversely, overexpression of HDAC4 repressed p21 promoter activity. p21 was likely a direct target of HDAC4, because HDAC4 down-regulation increased p21 mRNA when protein synthesis was inhibited by cycloheximide. The importance of p21 repression in HDAC4-mediated growth promotion was demonstrated by the failure of HDAC4 down-regulation to induce growth arrest in HCT116 p21-null cells. HDAC4 down-regulation failed to induce p21 when Sp1 was functionally inhibited by mithramycin or siRNA-mediated down-regulation. HDAC4 expression overlapped with that of Sp1, and a physical interaction was demonstrated by coimmunoprecipitation. Chromatin immunoprecipitation (ChIP) and sequential ChIP analyses demonstrated Sp1-dependent binding of HDAC4 to the proximal p21 promoter, likely directed through the HDAC4–HDAC3–N-CoR/SMRT corepressor complex. Consistent with increased transcription, HDAC4 or SMRT down-regulation resulted in increased histone H3 acetylation at the proximal p21 promoter locus. These studies identify HDAC4 as a novel regulator of colon cell proliferation through repression of p21.

hSgo2 (previously annotated as Tripin) was recently reported to be a new inner centromere protein that is essential for centromere cohesion (Kitajima et al., 2006). In this study, we show that hSgo2 exhibits a dynamic distribution pattern, and that its localization depends on the BUB1 and Aurora B kinases. hSgo2 is concentrated at the inner centromere of unattached kinetochores, but extends toward the kinetochores that are under tension. This localization pattern is reminiscent of MCAK, which is a microtubule depolymerase that is believed to be a key component of the error correction mechanism at kinetochores. Indeed, we found that hSgo2 is essential for MCAK to localize to the centromere. Delocalization of MCAK accounts for why cells depleted of hSgo2 exhibit kinetochore attachment defects that go uncorrected, despite a transient delay in the onset of anaphase. Consequently, these cells exhibit a high frequency of lagging chromosomes when they enter anaphase. We confirmed that hSgo2 is associated with PP2A, and we propose that it contributes to the spatial regulation of MCAK activity within inner centromere and kinetochore.

Pancreatic and colorectal carcinomas frequently express oncogenic/mutant K-Ras that contributes to both tumorigenesis and clinically observed resistance to radiation treatment. We have previously shown that farnesyltransferase inhibitors (FTI) radiosensitize many pancreatic and colorectal cancer cell lines that express oncogenic K-ras at doses that inhibit the prenylation and activation of H-Ras but not K-Ras. In the present study, we have examined the mechanism of FTI-mediated radiosensitization in cell lines that express oncogenic K-Ras and found that wild-type H-Ras is a contributor to radiation survival in tumor cells that express oncogenic K-Ras. In these experiments, inhibiting the expression of oncogenic K-Ras, wild-type H-Ras, or epidermal growth factor receptor (EGFR) led to similar levels of radiosensitization as treatment with the FTI tipifarnib. Treatment with the EGFR inhibitor gefitinib led to similar levels of radiosensitization, and the combinations of tipifarnib or gefitinib plus inhibition of K-Ras, H-Ras, or EGFR expression did not provide additional radiosensitization compared with tipifarnib or gefitinib alone. Finally, supplementing culture medium with the EGFR ligand transforming growth factor α was able to reverse the radiosensitizing effect of inhibiting K-ras expression. Taken together, these findings suggest that EGFR-activated H-Ras signaling is initiated by oncogenic K-Ras to promote radiation survival in pancreatic and colorectal cancers.

Long Interspersed Elements (LINE-1s, L1s) are the most active mobile elements in the human genome and account for a significant fraction of its mass. The propagation of L1 in the human genome requires disruption and repair of DNA at the site of integration. As Barbara McClintock first hypothesized, genotoxic stress may contribute to the mobilization of transposable elements, and conversely, element mobility may contribute to genotoxic stress. We tested the ability of genotoxic agents to increase L1 retrotransposition in a cultured cell assay. We observed that cells exposed to gamma radiation exhibited increased levels of L1 retrotransposition. The L1 retrotransposition frequency was proportional to the number of phosphorylated H2AX foci, an indicator of genotoxic stress. To explore the role of the L1 endonuclease in this context, endonuclease-deficient tagged L1 constructs were produced and tested for their activity in irradiated cells. The activity of the endonuclease-deficient L1 was very low in irradiated cells, suggesting that most L1 insertions in irradiated cells still use the L1 endonuclease. Consistent with this interpretation, DNA sequences that flank L1 insertions in irradiated cells harbored target site duplications. These results suggest that increased L1 retrotransposition in irradiated cells is endonuclease dependent. The mobilization of L1 in irradiated cells potentially contributes to genomic instability and could be a driving force for secondary mutations in patients undergoing radiation therapy.

The fidelity of chromosomal duplication is monitored by cell cycle checkpoints operational during mitosis. One such cell cycle delay is invoked by microtubule-targeting agents such as nocodazole or paclitaxel (Taxol) and is mediated by mitotic checkpoint proteins that include BubR1. Relatively little is known about the regulation of expression and stability of BubR1 (or other checkpoint proteins) and how these factors dictate the durability of the cell cycle delay. We report here that treatment of HeLa cells with spindle-disrupting agents resulted in caspase activation and precipitated the cleavage of BubR1. This mechanism ultimately leads to reduced levels of full-length protein, which are accompanied by abrogation of the mitotic block; the checkpoint abrogation is substantially accelerated by inhibition of de novo protein synthesis. In contrast, inhibition of caspase activity blocked BubR1 degradation and prolonged mitosis. To confirm a direct link between caspase activity and BubR1 protein expression, we identified by site-directed mutagenesis the specific caspase cleavage sites cleaved after exposure to paclitaxel. Surprisingly, BubR1 has two sites of cleavage: primarily at Asp607/Asp610 and secondarily at Asp576/Asp579. BubR1 mutated at both locations (BubR1Δ579Δ610) was resistant to paclitaxel-induced degradation. Expression of BubR1Δ579Δ610 augmented the mitotic delay induced by spindle disruption in transfected cells as well as in clones engineered to inducibly express the mutant protein upon exposure to doxycycline and ultimately led to increased aneuploidy. Underscoring the importance of these caspase cleavage sites, both tetrapeptide motifs are identified in the amino acid sequences of human, mouse, chicken, and Xenopus BubR1. These results are potentially the first to link the control of the stability of a key mitotic checkpoint protein to caspase activation, a regulatory pathway that may be involved in killing defective cells and that has been evolutionarily conserved.

DNA and histone synthesis are both triggered at the beginning of S phase by cyclin/cdk2 activity. Previous studies showed that inhibition of DNA synthesis with hydroxyurea or cytosine arabinoside (AraC) triggers a concerted repression of histone synthesis, indicating that sustained histone synthesis depends on continued DNA synthesis. Here we show that ectopic expression of HIRA, the likely human ortholog of two cell cycle-regulated repressors of histone gene transcription in yeast (Hir1p and Hir2p), represses transcription of histones and that this, in turn, triggers a concerted block of DNA synthesis. Thus, in mammalian cells sustained DNA synthesis and histone synthesis are mutually dependent on each other during S phase. Although cyclin/cdk2 activity drives activation of both DNA and histone synthesis at the G1/S transition of cycling cells, concerted repression of DNA or histone synthesis in response to inhibition of either one of these is not accompanied by prolonged inhibition of cyclin A/cdk2 or E/cdk2 activity. Therefore, during S phase coupling of DNA and histone synthesis occurs, at least in part, through a mechanism that is independent of cyclin/cdk2 activity. Coupling of DNA and histone synthesis in S phase presumably contributes to the prompt and orderly assembly of newly replicated DNA into chromatin.