Oxidative stress due to radiation in CD34(+) hematopoietic progenitor cells: protection by IGF-1.

Radiation exerts direct as well as indirect effects on DNA through the generation of reactive oxygen species (ROS). Irradiated hematopoietic progenitor cells (HPCs) experience DNA strand breaks, favoring genetic instability, due to ROS generation. Our aim was to study the effect of a range of radiation doses in HPCs and the possible protective mechanisms activated by insulin-like growth factor-1 (IGF-1). ROS generation was evaluated, in the presence or absence of IGF-1 in liquid cultures of human HPCs-CD34(+) irradiated with 1-, 2- and 5-Gy X-rays, using a flow cytometry assay. Manganese superoxide dismutase (MnSOD) expression was studied by western blot analysis and visualized by an immunofluorescence assay. Apoptosis was estimated using the following assays: Annexin-V assay, DNA degradation assay, BCL-2/BAX mRNA and protein levels and caspase-9 protein immunofluorescence visualization. Viability and clonogenic potential were studied in irradiated HPCs. The generation of superoxide anion radicals at an early and a late time point was increased, while the hydrogen peroxide generation at a late time point was stable. IGF-1 presence further enhanced the radiation-induced increase of MnSOD at 24 h post irradiation. IGF-1 inhibited the mitochondria-mediated pathway of apoptosis by regulating the m-RNA and protein expression of BAX, BCL-2 and the BCL-2/BAX ratio and by decreasing caspase-9 protein expression. IGF-1 presence in culture media of irradiated cells restored the clonogenic capacity and the viability of HPCs as well. In conclusion, IGF-1 protects HPCs-CD34(+) from radiation effects, by eliminating the oxidative microenvironment through the enhancement of MnSOD activation and by regulating the mitochondria-mediated pathway of apoptosis.

The survivin -31 snp in human colorectal cancer correlates with survivin splice variant expression and improved overall survival.

BACKGROUND: Survivin is involved in the regulation of cell division and survival, two key processes in cancer. The majority of studies on survivin in colorectal cancer (CRC) have focused on protein expression and less is known about the expression of survivin splicing variants or survivin gene polymorphisms in CRC. In the present study, the mRNA levels of the five known isoforms of survivin as well as survivin protein were assessed in matched normal and neoplastic colorectal tissue. Moreover, the 9386 C/T and -31 G/C polymorphisms were investigated. METHODS: Quantitative RT-PCR was used to assess mRNA levels in fresh/frozen tissue samples. Protein levels were immunohistochemically evaluated on formalin-fixed paraffin-embedded tissue sections. Individuals were genotyped using real time PCR. RESULTS: Expression of all 5 survivin splice variants as well as survivin protein was elevated in colorectal carcinomas compared to normal tissue. Specific splice variant expression differentially correlated with clinicopathological parameters. Furthermore, both snps correlated with splice variant levels or their ratios in colorectal carcinomas while the -31 G/C snp may be related to CRC development and improved overall survival. CONCLUSION: Our results support a role of survivin in colorectal carcinogenesis while the -31 G/C snp may constitute a marker of survival.

The survivin -31 snp in human colorectal cancer correlates with survivin splice variant expression and improved overall survival.

BACKGROUND: survivin is involved in the regulation of cell division and survival, two key processes in cancer. The majority of studies on survivin in colorectal cancer (CRC) have focused on protein expression and less is known about the expression of survivin splicing variants or survivin gene polymorphisms in CRC. In the present study, the mRNA levels of the five known isoforms of survivin as well as survivin protein were assessed in matched normal and neoplastic colorectal tissue. Moreover, the 9386C/T and -31G/C polymorphisms were investigated. METHODS: quantitative RT-PCR was used to assess mRNA levels in fresh/frozen tissue samples. Protein levels were immunohistochemically evaluated on formalin-fixed paraffin-embedded tissue sections. Individuals were genotyped using real time PCR. RESULTS: expression of all 5 survivin splice variants as well as survivin protein was elevated in colorectal carcinomas compared to normal tissue. Specific splice variant expression differentially correlated with clinicopathological parameters. Furthermore, both snps correlated with splice variant levels or their ratios in colorectal carcinomas while the -31G/C snp may be related to CRC development and improved overall survival. CONCLUSION: our results support a role of survivin in colorectal carcinogenesis while the -31G/C snp may constitute a marker of survival.

TGF-beta repressors SnoN and Ski are implicated in human colorectal carcinogenesis.

BACKGROUND: The TGF-beta signaling repressors SnoN and Ski have been critically implicated in human cancer. METHODS: To explore the role of SnoN and Ski in the development and progression of colorectal cancer we examined their protein expression profile by immunohistochemistry in a series of human colorectal adenomas, carcinomas and lymph node metastases. The mRNA expression of SnoN was also quantified by Real-Time RT-PCR. RESULTS: SnoN and Ski were overexpressed both in adenomas with severe dysplasia and colorectal carcinomas. Protein expression was cytoplasmic and nuclear with predominant cytoplasmic localization. The subcellular localization was related differently to pathologic variables of colorectal carcinomas. Although there was no significant association of protein levels with tumor invasion and metastasis, a significant correlation of nuclear SnoN and Ski with beta-catenin pathway was observed. Moreover, SnoN mRNA did not differ in carcinomas as compared to normal control and there was no correlation between SnoN protein and mRNA levels. CONCLUSION: Our findings suggest that SnoN and Ski exert oncogenic effects in human colorectal carcinogenesis and their overexpression is implicated in early stage disease.

Dual targeting of EGFR and HER-2 in colon cancer cell lines.

PURPOSE: A number of studies have revealed that coexpression of EGFR and HER-2 has been found in a subset of colon cancers and may cooperatively promote tumor cell growth and survival. In the present work, two tyrosine kinase inhibitors, gefitinib and lapatinib, together with trastuzumab, raised a monoclonal antibody against HER-2 were evaluated in two colon cancer cell lines, DLD-1 and Caco-2. The aim of the study was to investigate their effect on tumor cell proliferation and apoptosis. METHODS: Cell proliferation was assessed using the MTT assay and apoptosis was evaluated by DNA fragmentation and the Annexin V binding assay. EGFR and HER-2 protein and mRNA levels were evaluated by immunoblotting and quantitative RT-PCR, respectively. RESULTS: Treatment of cells with each agent alone resulted in inhibition of cell proliferation after 48 h in a dose-dependent manner except for trastuzumab, which did not alter cell proliferation of DLD-1. Apoptosis increased in DLD-1 cells, after 24 h treatment with gefitinib. None of the tested agents altered apoptosis in Caco-2 cells. HER-2 and EGFR protein levels did not follow the changes of mRNA levels after treatment with the tested agents. CONCLUSIONS: Tauhe inhibitory effect of these agents on cell proliferation and the induction of apoptosis differ for the two colon cancer cell lines under consideration. Further studies are necessary to investigate the way they exert their antitumor effect.

Abdominal radiation initiates apoptotic mechanism in rat femur bone marrow cells in vivo that is reversed by IGF-1 administration.

PURPOSE: Radiation induces apoptosis as a result of damage to cellular DNA and RNA. The aim of our work was to study the effect of radiation on rat bone marrow cells (as a neighboring tissue) in the context of a model of experimental radiation enteritis in rats. The effect of systematic administration in irradiated animals of r-IGF-1 and GH was also studied. MATERIALS AND METHODS: Wistar type, normal rats, were divided in 4 groups. One control group and the other 3 groups were irradiated in the abdomen. The measured scattered irradiation in the femur ranged from 16.5 to 47.3 cGy. In 2 groups of irradiated animals, rIGF-1 (0.1 microg/g of body weight twice/d) and rGH (0.25 microg/g of body weight /d) were administered. Bone marrow cells were harvested from both femurs. DNA and RNA were analyzed in specific gels. The m-RNA was hybridized for c-fos proto-oncogene expression. RESULTS: The calculated low dose of radiation that affected the femurs of the animals induced reduction in bone marrow cell numbers and endonuclease activation manifested by subsequent fragmentation of DNA and RNA. This phenomenon was reversed by rGH and rIGF-1 administration. The c-fos proto-oncogene expression was upregulated by irradiation. CONCLUSION: These observations indicate that scattered low dose radiation is capable of initiating apoptosis in rat bone marrow cells and rGH and rIGF-1 administration reverse this process.