Burong Hu

Unrepaired clustered DNA lesions induce chromosome breakage in human cells

Clustered DNA damage induced by ionizing radiation is refractory to repair and may trigger carcinogenic events for reasons that are not well understood. Here, we used an in situ method to directly monitor induction and repair of clustered DNA lesions in individual cells. We showed, consistent with biophysical modeling, that the kinetics of loss of clustered DNA lesions was substantially compromised in human fibroblasts. The unique spatial distribution of different types of DNA lesions within the clustered damages, but not the physical location of these damages within the subnuclear domains, determined the cellular ability to repair the damage. We then examined checkpoint arrest mechanisms and yield of gross chromosomal aberrations. Induction of nonrepairable clustered damage affected only G2 accumulation but not the early G2/M checkpoint. Further, cells that were released from the G2/M checkpoint with unrepaired clustered damage manifested a spectrum of chromosome aberrations in mitosis. Difficulties associated with clustered DNA damage repair and checkpoint release before the completion of clustered DNA damage repair appear to promote genome instability that may lead to carcinogenesis.

Effects of ionizing radiation on three-dimensional human vessel models: differential effects according to radiation quality and cellular development.

Abstract Little is known about the effects of space radiation on the human body. There are a number of potential chronic and acute effects, and one major target for noncarcinogenic effects is the human vasculature. Cellular stress, inflammatory response, and other radiation effects on endothelial cells may affect vascular function. This study was aimed at understanding the effects of space ionizing radiation on the formation and maintenance of capillary-like blood vessels. We used a 3D human vessel model created with human endothelial cells in a gel matrix to assess the effects of low-LET protons and high-LET iron ions. Iron ions were more damaging and caused significant reduction in the length of intact vessels in both developing and mature vessels at a dose of 80 cGy. Protons had no effect on mature vessels up to a dose of 3.2 Gy but did inhibit vessel formation at 80 cGy. Comparison with γ radiation showed that photons had even less effect, although, as with protons, developing vessels were more sensitive. Apoptosis assays showed that inhibition of vessel development or deterioration of mature vessels was not due to cell death by apoptosis even in the case of iron ions. These are the first data to show the effects of radiation with varying linear energy transfer on a human vessel model.

In Situ Visualization of DSBs to Assess the Extranuclear/Extracellular Effects Induced by Low-Dose α-Particle Irradiation

Abstract Hu, B., Han, W., Wu, L., Feng, H., Liu, X., Zhang, L., Xu, A., Hei, T K. and Yu, Z. In Situ Visualization of DSBs to Assess the Extranuclear/Extracellular Effects Induced by Low-Dose α-Particle Irradiation. Radiat. Res. 164, 286–291 (2005). Extranuclear/extracellular effects may have a significant effect on low-dose radiation risk assessment as well as on the shape of the dose–response relationship. Numerous studies using different end points such as sister chromatid exchanges, micronuclei and mutation have shown that this phenomenon exists in many cell types. However, these end points mostly reflect the late events after radiation damage, and little is known about the early response in this phenomenon. DNA double-strand breaks (DSBs) induced by ionizing radiation or carcinogenic chemicals can be visualized in situ using γ-H2AX immunofluorescence staining, and there is evidence that the number of γ-H2AX foci can be closely correlated with DSBs induced. Here we used γ-H2AX as a biomarker to assess the extranuclear/extracellular effects induced by low-dose α particles in situ. The results show that a greater fraction of positive cells with DSBs (48.6%) was observed than the number of cells whose nuclei were actually traversed by the 1-cGy dose of α particles (9.2%). The fraction of DSB-positive cells was greatly reduced after treatment with either lindane or DMSO. These results suggest that in situ visualization of DSBs can be used to assess radiation-induced extranuclear/extracellular effects soon after irradiation. Moreover, the in situ DSB assay may provide a means to evaluate the spatial effect on unirradiated cells that are located in the neighboring region of cells irradiated by α particles.

Irreparable complex DNA double-strand breaks induce chromosome breakage in organotypic three-dimensional human lung epithelial cell culture

DNA damage and consequent mutations initiate the multistep carcinogenic process. Differentiated cells have a reduced capacity to repair DNA lesions, but the biological impact of unrepaired DNA lesions in differentiated lung epithelial cells is unclear. Here, we used a novel organotypic human lung three-dimensional (3D) model to investigate the biological significance of unrepaired DNA lesions in differentiated lung epithelial cells. We showed, consistent with existing notions that the kinetics of loss of simple double-strand breaks (DSBs) were significantly reduced in organotypic 3D culture compared to kinetics of repair in two-dimensional (2D) culture. Strikingly, we found that, unlike simple DSBs, a majority of complex DNA lesions were irreparable in organotypic 3D culture. Levels of expression of multiple DNA damage repair pathway genes were significantly reduced in the organotypic 3D culture compared with those in 2D culture providing molecular evidence for the defective DNA damage repair in organotypic culture. Further, when differentiated cells with unrepaired DNA lesions re-entered the cell cycle, they manifested a spectrum of gross-chromosomal aberrations in mitosis. Our data suggest that downregulation of multiple DNA repair pathway genes in differentiated cells renders them vulnerable to DSBs, promoting genome instability that may lead to carcinogenesis.

Transforming growth factor-β-induced protein (TGFBI) suppresses mesothelioma progression through the Akt/mTOR pathway

As an uncommon cancer, mesothelioma is very hard to treat with a low average survival rate owing to its usual late detection and being highly invasive. The link between asbestos exposure and the development of mesothelioma in humans is unequivocal. TGFBI, a secreted protein that is induced by transforming growth factor-β in various human cell types, has been shown to be associated with tumorigenesis in various types of tumors. It has been demonstrated that TGFBI expression is markedly suppressed in asbestos-induced tumorigenic cells, while an ectopic expression of TGFBI significantly suppresses tumorigenicity and progression in human bronchial epithelial cells. In order to delineate a potential role of TGFBI in mediating the molecular events that occur in mesothelioma tumorigenesis, we generated stable TGFBI knockdown mutants from the mesothelium cell line Met-5A by using an shRNA approach, and secondly created ectopic TGFBI overexpression mutants from the mesothelioma cell line H28 in which TGFBI is absent. We observed that in the absence of TGFBI, the knockdown mesothelial and mesothelioma cell lines exhibited an elevated proliferation rate, enhanced plating efficiency, increased anchorage-independent growth, as well as an increased cellular protein synthesis rate as compared with their respective controls. Furthermore, cell cycle regulatory proteins c-myc/cyclin D1/phosphor-Rb were upregulated; a more active PI3K/Akt/mTOR signaling pathway was also detected in TGFBI-depleted cell lines. These findings suggest that TGFBI may repress mesothelioma tumorigenesis and progression via the PI3K/Akt signaling pathway.

Synthesis and structure-activity relationships of N-methyl-5,6,7-trimethoxylindoles as novel antimitotic and vascular disrupting agents.

Several new series of 5,6,7-trimethoxyindole derivatives were synthesized and their structure-activity relationships (SARs) were studied. Some of these compounds exhibited strong antiproliferative activities in the submicromolar range. N-Methyl-5,6,7-trimethoxylindoles 21 and 31 displayed the highest antiproliferative activities, with IC50 values ranging from 22 to 125 nM in four human cancer cell lines and activated human umbilical vein endothelial cells (HUVECs). In addition to vascular disrupting activity verified by in vitro assays, compounds 21 and 31 displayed much higher selectivity for activated HUVECs versus quiescent HUVECs than those of colchicine and combretastatinA-4. The polymerization of cancer cell tubulin was inhibited and the cell cycle was arrested in the G2/M phase after treatment with 21 and 31. It was showed that 21 disrupted tumor vasculature by use of in vivo assay. Our results suggest that these two new compounds we synthesized may become the promising leads for the development of vascular disrupting agents.

Intrachromosomal Changes and Genomic Instability in Site-Specific Microbeam-Irradiated and Bystander Human-Hamster Hybrid Cells

Exposure to ionizing radiation may induce a heritable genomic instability phenotype that results in a persisting and enhanced genetic and functional change among the progeny of irradiated cells. Since radiation-induced bystander effects have been demonstrated with a variety of biological end points under both in vitro and in vivo conditions, this raises the question whether cytoplasmic irradiation or the radiation-induced bystander effect can also lead to delayed genomic instability. In the present study, we used the Radiological Research Accelerator Facility charged-particle microbeam for precise nuclear or cytoplasmic irradiation. The progeny of irradiated and the bystander human hamster hybrid (AL) cells were analyzed using multicolor banding (mBAND) to examine persistent chromosomal changes. Our results showed that the numbers of metaphase cells involving changes of human chromosome 11 (including rearrangement, deletion and duplication) were significantly higher than that of the control in the progeny of both nuclear and cytoplasmic targeted cells. These chromosomal changes could also be detected among the progeny of bystander cells. mBAND analyses of clonal isolates from nuclear and cytoplasm irradiations as well as the bystander cell group showed that chromosomal unstable clones were generated. Analyses of clonal stability after long-term culture indicated no significant change in the number of unstable clones for the duration of culture in each irradiated group. These results suggest that genomic instability that is manifested after ionizing radiation exposure is not dependent on direct damage to the cell nucleus.

Protein kinase C epsilon is involved in ionizing radiation induced bystander response in human cells.

Our earlier study demonstrated the induction of PKC isoforms (betaII, PKC-alpha/beta, PKC-theta) by ionizing radiation induced bystander response in human cells. In this study, we extended our investigation to yet another important member of PKC family, PKC epsilon (PKCepsilon). PKCepsilon functions both as an anti-apoptotic and pro-apoptotic protein and it is the only PKC isozyme implicated in oncogenesis. Given the importance of PKCepsilon in oncogenesis, we wished to determine whether or not PKCepsilon is involved in bystander response. Gene expression array analysis demonstrated a 2-3-fold increase in PKCepsilon expression in the bystander human primary fibroblast cells that were co-cultured in double-sided Mylar dishes for 3h with human primary fibroblast cells irradiated with 5Gy of alpha-particles. The elevated PKCepsilon expression in bystander cells was verified by quantitative real time PCR. Suppression of PKCepsilon expression by small molecule inhibitor Bisindolylmaleimide IX (Ro 31-8220) considerably reduced the frequency of micronuclei (MN) induced both by 5Gy of gamma-rays (low LET) and alpha-particles (high LET) in bystander cells. Similar cytoprotective effects were observed in bystander cells after siRNA mediated silencing of PKCepsilon suggestive of its critical role in mediating some of the bystander effects (BE). Our novel study suggests the possibility that PKC signaling pathway may be a critical molecular target for suppression of ionizing radiation induced biological effects in bystander cells.

Reprogramming mediated radio-resistance of 3D-grown cancer cells

In vitro 3D growth of tumors is a new cell culture model that more closely mimics the features of the in vivo environment and is being used increasingly in the field of biological and medical research. It has been demonstrated that cancer cells cultured in 3D matrices are more radio-resistant compared with cells in monolayers. However, the mechanisms causing this difference remain unclear. Here we show that cancer cells cultured in a 3D microenvironment demonstrated an increase in cells with stem cell properties. This was confirmed by the finding that cells in 3D cultures upregulated the gene and protein expression of the stem cell reprogramming factors such as OCT4, SOX2, NANOG, LIN28 and miR-302a, compared with cells in monolayers. Moreover, the expression of β-catenin, a regulating molecule of reprogramming factors, also increased in 3D-grown cancer cells. These findings suggest that cancer cells were reprogrammed to become stem cell–like cancer cells in a 3D growth culture microenvironment. Since cancer stem cell–like cells demonstrate an increased radio-resistance and chemo-resistance, our results offer a new perspective as to why. Our findings shed new light on understanding the features of the 3D growth cell model and its application in basic research into clinical radiotherapy and medicine.

FANCD2 influences replication fork processes and genome stability in response to clustered DSBs

Fanconi Anemia (FA) is a cancer predisposition syndrome and the factors defective in FA are involved in DNA replication, DNA damage repair and tumor suppression. Here, we show that FANCD2 is critical for genome stability maintenance in response to high-linear energy transfer (LET) radiation. We found that FANCD2 is monoubiquitinated and recruited to the sites of clustered DNA double-stranded breaks (DSBs) specifically in S/G2 cells after high-LET radiation. Further, FANCD2 facilitated the repair of clustered DSBs in S/G2 cells and proper progression of S-phase. Furthermore, lack of FANCD2 led to a reduced rate of replication fork progression and elevated levels of both replication fork stalling and new origin firing in response to high-LET radiation. Mechanistically, FANCD2 is required for correct recruitment of RPA2 and Rad51 to the sites of clustered DSBs and that is critical for proper processing of clustered DSBs. Significantly, FANCD2-decifient cells exhibited defective chromosome segregation, elevated levels of chromosomal aberrations, and anchorage-independent growth in response to high-LET radiation. These findings establish FANCD2 as a key factor in genome stability maintenance in response to high-LET radiation and as a promising target to improve cancer therapy.