Vicenin-2: a potential radiosensitizer of non-small cell lung cancer cells.

Non-small cell lung cancer (NSCLC) is a major form of cancer and is resistant to chemo- and radio-therapy. Vicenin-2 (VCN-2) is a flavonoid obtained from Ocimum sanctum L. and it has been reported to have radioprotective and anti-cancer properties. This study was conducted to check for the radiosensitizing potential of VCN-2 in the NSCLC cell line, NCI-H23. NCI-H23 cells were exposed to VCN-2 singularly, and to X-rays with and without prior VCN-2 treatment. Cytotoxicity assay, cell proliferation assay, caspase-3 activity assay, DNA fragmentation assay and Western blotting for Rad50, MMP-2 and p21 were performed to investigate the radiosensitizing properties of VCN-2. Fibroblast survival assay was performed using HEK293T cells to check for any adverse effects of VCN-2 on normal fibroblast cell line. VCN-2 singularly and in combination with radiation reduced the surviving cancer cells, increased caspase-3 activity, increased DNA fragmentation, increased the levels of Rad50 and lowered levels of MMP-2 and p21 proteins while being non-toxic and radioprotective to the fibroblast cells. VCN-2 showed a potent radiosensitizing property while also showing a chemotherapeutic property against NSCLC cell line NCI-H23.

Critical role of RecA and RecF proteins in strand break rejoining and maintenance of fidelity of rejoining following gamma-radiation-induced damage to pMTa4 DNA in E. coli.

PURPOSE: This study was undertaken to understand the roles of RecA and RecF proteins in strand break rejoining and maintenance of fidelity of the process following exposure of E. coli to gamma-radiation in vivo. MATERIALS AND METHODS: A plasmid DNA construct, pMTa4, was transformed into isogenic repair proficient (wild) and deficient (recF and recA) E. coli strains and gamma-irradiated up to 30 Gy in vivo. The plasmid DNA was isolated under repair non-permissive (R-)and permissive (R+) conditions and analyzed by gel electrophoresis for the yields of single strand breaks (SSB) and double strand breaks (DSB) and their repair. The clonogenic survival of the E. coli was also recorded. The effects of gamma-irradiation on recA reconstituted with cell free extract of wild strain or ultra-violet (UV)-irradiation were also monitored. RESULTS: None of the strains used in this investigation showed effects of radiation-induced oxidative base damage. The dose dependent increase in SSB and DSB on pMTa4 in wild and recF mutants in R- condition were abolished upon repair incubation. The recA mutant exhibited a disturbed yield of SSB and DSB along with formation of gamma-radiation-induced 'ladder'. The 'ladder' was not observed after repair incubation, UV-irradiation or gamma-irradiation in presence of cell-free extract of wild strain. The survival of recA mutants was seriously compromised. CONCLUSIONS: Wild, recF and recA strains of E. coli could repair gamma-irradiation-induced oxidative damage to base or nucleotide (NT) in vivo. In absence of either RecA or RecF proteins, efficiency of rejoining of strand went down; RecA proteins seemed more critical than RecF in this. High fidelity or correct rejoining of strand breaks, on the other hand, seemed to require simultaneous presence of both RecA and RecF proteins.

Progressive reduction in poly-ADP-ribosylation of histone proteins during Dalton's lymphoma induced ascites tumorigenesis in mice.

Poly-ADP-ribosylation (PAR) reaction, which primarily modifies histone proteins, is postulated to have decisive influence in carcinogenesis. Therefore, it has been suggested as a biomarker of carcinogenesis. Dalton's lymphoma induced ascites tumorigenesis in mice provides a convenient model to study PAR of histone proteins during late stage of cancer development. Using a Western blot immunoprobe assay of cellular PAR, this report shows that PAR of liver and spleen histone proteins was progressively and significantly reduced during Dalton's lymphoma induced ascites tumorigenesis in mice. Implication of the findings in early detection of cancer has been discussed.

Influence of histone acetylation on the modification of cytoplasmic and nuclear proteins by ADP-ribosylation in response to free radicals.

Inhibition of histone deacetylase by addition of 5 mM n-sodium butyrate to the growth medium increases the utilization of [32P]NAD+ and ADP-ribosylation (ADPR) of total cellular proteins of V79, HeLa, mouse B16, mouse Fib/T and human T1 kidney cells by a factor of 1.2-2.3. When the ADP-ribosylase is challenged by exposing cells to damage by .OH radicals (25 microM CuSO4 2.8 mM H2O2) ADPR increases by factors of 5.7-6.0 and 3.2-4.0 in normal and butyrated cells, respectively. Operation of the free radical generator is supported by the response to EDTA and radical scavengers. Densitometric analysis of autoradiographs from SDS-gels show that butyrate exposure increases basal ADPR-modification of histones from T1 cells by factors of 1.1-1.9. Addition of .OH radicals increases the ADPR modifications of histones 4.4-8.7-fold in normal cells and 3.2-6.7-fold in butyrate exposed cells. Butyrate exposure elevates base level ADPR-modification and reduces subsequent ADPR-modification initiated by DNA damage. The results are consistent with the view that ADPR-modification and histone acetylation have overlapping functions and probably induce similar structural changes in chromatin.

Differential DNA strand breaking abilities of *OH and ROS generating radiomimetic chemicals and gamma-rays: study of plasmid DNA, pMTa4, in vitro.

Reactive oxygen species (ROS) interact with components of a living cell. Among them *OH is known to cause major oxidative damage to living cells and is proposed to be involved in pathogenesis including carcinogenesis. Proper understanding of consequences of such damage is, therefore, medically relevant. In this report, aqueous solution of plasmid DNA, pMTa4, has been exposed to Fenton oxidant and Haber-Weiss oxidant mediated free radical generating chemical systems, and 60Co gamma-rays in vitro either alone or in combination to study their strand breaking abilities. The exposed pMTa4 was analyzed by agarose gel electrophoresis. The results show qualitative differences in the induction of strand breaks on the plasmid pMTa4 molecule by the iron (Fe2+), copper (Cu2+) or gamma-rays mediated *OH and other ROS.

UV-C radiation induced conformational relaxation of pMTa4 DNA in Escherichia coli may be the cause of single strand breaks.

PURPOSE: The biological consequences of initial physicochemical events following exposure of DNA to germicidal (254 nm) ultraviolet C (UV-C) radiation are not fully understood despite progress that has been made. In particular the cause of UV-C induced single strand breaks is not known. This question has been addressed in the present investigation. MATERIALS AND METHODS: A plasmid construct, pMTa4, was exposed to UV-C in vitro as well as in vivo after transforming the plasmid into a repair proficient wild type and repair deficient, recF, mutant of E. coli. Following UV exposure in vivo, the plasmid was isolated under repair non-permissive and permissive conditions. The plasmid isolate and the pure super-coiled closed circular (CC) topological form of the plasmid were analyzed by agarose gel electrophoresis. The dependence of UV-C induced damage and conformational changes on the dose of radiation as well as on the duration of post-irradiation repair incubations was observed. The influence of UV-C on hyperchromic change and intercalation of ethidium bromide into plasmid DNA were also recorded. RESULTS: UV-C exposure of pMTa4 DNA in vitro and in vivo induced dose dependent, but sparsely placed, single strand breaks (SSB). While the wild type (AB1157) E. coli was able to repair SSB nearly completely under repair permissive condition, the recF (JC9239) mutant failed to do so. A dose-dependent relaxation of super-structure of CC form of pMTa4 DNA concomitant with enhanced ethidium bromide intercalation into the plasmid DNA was observed. CONCLUSION: It is proposed that the conformational relaxation generated negative super-coiling strain on the DNA backbone of CC form of plasmid as well as exposed chemical bonds for hydrolytic cleavage. This might be the cause of the production of sparsely placed single strand breaks in pMTa4 upon exposure to low doses of UV-C.

Evaluation of endogenous control gene(s) for gene expression studies in human blood exposed to 60Co γ-rays ex vivo.

In gene expression studies, it is critical to normalize data using a stably expressed endogenous control gene in order to obtain accurate and reliable results. However, we currently do not have a universally applied endogenous control gene for normalization of data for gene expression studies, particularly those involving (60)Co γ-ray-exposed human blood samples. In this study, a comparative assessment of the gene expression of six widely used housekeeping endogenous control genes, namely 18S, ACTB, B2M, GAPDH, MT-ATP6 and CDKN1A, was undertaken for a range of (60)Co γ-ray doses (0.5, 1.0, 2.0 and 4.0 Gy) at 8.4 Gy min(-1) at 0 and 24 h post-irradiation time intervals. Using the NormFinder algorithm, real-time PCR data obtained from six individuals (three males and three females) were analyzed with respect to the threshold cycle (Ct) value and abundance, ΔCt pair-wise comparison, intra- and inter-group variability assessments, etc. GAPDH, either alone or in combination with 18S, was found to be the most suitable endogenous control gene and should be used in gene expression studies, especially those involving qPCR of γ-ray-exposed human blood samples.

Consensus reference gene(s) for gene expression studies in human cancers: end of the tunnel visible?

BACKGROUND: Gene expression studies are increasingly used to provide valuable information on the diagnosis and prognosis of human cancers. Also, for in vitro and in vivo experimental cancer models gene expression studies are widely used. The complex algorithms of differential gene expression analyses require normalization of data against a reference or normalizer gene, or a set of such genes. For this purpose, mostly invariant housekeeping genes are used. Unfortunately, however, there are no consensus (housekeeping) genes that serve as reference or normalizer for different human cancers. In fact, scientists have employed a wide range of reference genes across different types of cancer for normalization of gene expression data. As a consequence, comparisons of these data and/or data harmonizations are difficult to perform and challenging. In addition, an inadequate choice for a reference gene may obscure genuine changes and/or result in erroneous gene expression data comparisons. METHODS: In our effort to highlight the importance of selecting the most appropriate reference gene(s), we have screened the literature for gene expression studies published since the turn of the century on thirteen of the most prevalent human cancers worldwide. CONCLUSIONS: Based on the analysis of the data at hand, we firstly recommend that in each study the suitability of candidate reference gene(s) should carefully be evaluated in order to yield reliable differential gene expression data. Secondly, we recommend that a combination of PPIA and either GAPDH, ACTB, HPRT and TBP, or appropriate combinations of two or three of these genes, should be employed in future studies, to ensure that results from different studies on different human cancers can be harmonized. This approach will ultimately increase the depth of our understanding of gene expression signatures across human cancers.

Arecoline-induced changes of poly-ADP-ribosylation of cellular proteins and its influence on chromatin organization.

Arecoline, the major alkaloid of betel nut (Areca catechu L.) and a suspected carcinogen, has been implicated in human cancers of various sites. A considerable portion of the world's population is constantly exposed to arecoline due to the habit of masticating betel nuts. The present work relates to the study of early molecular events following chronic arecoline exposure at a dose of 10 microg/ml to Swiss albino mice. Poly-ADP-ribosylation of all cellular proteins, histones and poly-ADP-ribose polymerase were studied in bone marrow and spleen cells and correlated with the organizational status of the chromatin. While most proteins showed lowering of their poly-ADP-ribosylation following arecoline treatment, only histone protein H1 in spleen cells and H2B in bone marrow cells exhibited an increase. The chromatin of both the tissues was progressively relaxed upon arecoline exposure. The implications of these changes have been discussed regarding the process of initiation of arecoline-induced carcinogenesis.

Aqueous extract of betel-nut of north-east India induces DNA-strand breaks and enhances rate of cell proliferation in vitro. Effects of betel-nut extract in vitro.

An aqueous extract of betel-nut has been found to be able to induce strand breaks in DNA of mouse kidney cells in vitro. It has been also found to be able to enhance the rate of cell division at a dose of 100 micrograms/ml while a higher dose of 250 micrograms/ml was extremely toxic to the cells. Compared with arecoline (10 micrograms/ml), the aqueous extract of betel-nut seems to be a more potent carcinogen to mouse kidney cells in vitro.

Low dose exposure of diethylnitrosamine affects mice liver thymidine kinase.

Swiss albino mice exposed to 5 and 10 mg diethylnitrosamine kg-1 body weight by intravenous route up to four weeks showed cyto- and genotoxic effects. Distortion of cell and nucleus shapes and extensive necrosis were observed. Thymidine kinase activity in the liver declined in diethylnitrosamine dose and duration dependent manners. The adult-form of thymidine kinase isozyme declined continuously during this period. Simultaneously, two isozymic forms of thymidine kinase, with small anodic migrations in an electrophoretic field, were gradually induced. Significance of theses changes in diethylnitrosamine induced precarcinogenic toxicity has been discussed.

Response of high mobility group proteins of human kidney T1 and murine L 929 cell lines to heat shock.

High mobility group (HMG) proteins in human kidney T1 and murine L 929 cells have been investigated after exposure to heat shock at 41 degrees C and their influence on the organizational change of chromatin under heat shock condition has been examined. Results reveal that the two cell lines show differential response of the HMG proteins 1 & 2 and 14 & 17 to heat shock. Neither T1 nor L 929 cells show significant differences in response to heat shock with respect to the binding affinities of HMG proteins 1 & 2 or 14 & 17 to DNA, as revealed by DNase I sensitivity and chromatin reconstitution assays. Furthermore, the HMG proteins of both the non-heat shocked and the heat shocked T1 and L 929 cells can recover their chromatin activity following reconstitution. These findings suggest that although the HMG proteins might undergo some change in response to heat shock, their inherent potential of reassociation with DNA is still retained.

Study of unscheduled DNA synthesis following exposure of human cells to arecoline and extracts of betel nut in vitro.

Aqueous, acetic acid, hydrochloric acid and ethanol extracts of betel nut (Areca catechu L.) have been found to induce unscheduled DNA synthesis in Hep 2 cells obtained from human larynx carcinoma, in vitro. Different concentrations of extracts of betel nut induced dose-dependent unscheduled DNA synthesis in Hep 2 cells. Together with the viability of the Hep 2 cells, our results indicate that the aqueous and acetic acid extracts of betel nut induce relatively more unscheduled DNA synthesis than the hydrochloric acid and ethanol extracts and arecoline. The carcinogenic potency of raw and unprocessed betel nut of North-East India used in this study is discussed.

2-Mercaptopropionylglycine affords enhanced radioprotection after a liposome encapsulation.

Use of radioprotective drugs in radiotherapy is desirable to protect normal tissues. 2-mercaptopropionylglycine (MPG) has shown promising results in experimental radioprotection. In this report, a liposome drug delivery system for MPG has been used in Swiss albino mice exposed to 1 to 8 Gy whole body Gamma-irradiation to test whether or not this modality enhances the MPG afforded radioprotection. A statistically significant, dose dependent enhancement of protection by liposome encapsulated MPG (LEM) was observed. LEM, as compared to free MPG, improved the viabilities of spleen and bone marrow cells by factors between 1.11 and 2.23 for different doses of radiation.

Role of mouse spleen cell HMG proteins and their poly-ADP-ribosylation in betelnut induced carcinogenesis.

The role of high mobility group (HMG) proteins and their poly-ADP-ribosylation (PAR) in betel nut induced initiation of carcinogenesis in mice has been studied. A known carcinogen, diethylnitrosamine (DEN) was used as a positive control. Swiss albino mice were chronically exposed to aqueous extract of betel nut (AEBN) or DEN at low doses for up to 4 weeks. The poly-ADP-ribosylation (PAR) of spleen cell HMG proteins was monitored using [32P]-NAD+. Parallel to this, chromatin was subjected to DNase I cleavage and the organizational state of the chromatin was monitored. The PAR of HMG proteins showed a marked progressive reduction at different times following AEBN- or DEN treatment. HMG proteins isolated from the control and carcinogen treated mice were allowed to reassociate with the untreated spleen cells chromatin. The reassociated chromatin showed progressive relaxation in its superstructure. The results suggest that under the influence of potential carcinogens AEBN or DEN, the mouse spleen cell HMG proteins created molecular conditions favourable to initiation of cancer.

Change of ADP-ribosylation in human kidney T1-cells by various external stimuli.

Human kidney T1-cells in culture respond to various externally applied stresses, such as mechanical manipulations, trypsinization combined with agitation and centrifugation, Gamma-irradiation, and hyperthermia, by increasing the ADP-ribosylation (ADPR) of proteins in cytoplasm and nuclei. On the other hand, a strong static magnetic field or gravitational force reduces ADPR of proteins. These responses are easily detected in cells in monolayer, yet are masked when the conventional assay of ADPR is used on cells in suspension. The multitude of stimuli for either increasing or depressing ADPR of proteins including histones suggests a common pathway of triggering ADPR in which cellular membranes may be involved.

Neutrons affect ADP-ribosylation of proteins in human kidney T1-cells in vitro.

ADP-ribosylation (ADPR) of proteins has been shown to be involved with a variety of cellular responses in which chromatin organization and functions are affected. In order to look into this response, human kidney T1-cells were exposed in vitro to various doses up to 3 Gy of 6 MeV neutrons and compared with the effect caused by gamma photons. Whereas in case of neutrons the maximal inhibition of ADPR was reversed at 0.37 Gy, that in case of gamma-rays occurred at 1.5 Gy. For the reversal of inhibition of ADPR of proteins in T1-cells, neutrons were about 4-fold more efficient as compared to gamma rays.

Poly-ADP-ribosylation of histone proteins of human kidney T1-cells in vitro following gamma-irradiation.

Poly-ADP-ribosylation of cellular proteins is involved with radiation induced damage and its repair. It has been observed that suspension of human kidney T1-cells in vitro attained elevated levels of poly-ADP-ribosylation due to experimental manipulations necessary for preparation of single cell suspension from monolayer cell cultures. These cells in suspension were exposed to various doses of gamma-rays with or without subsequent repair incubation. The PADPR of histones H3, H1 and H2B increased with increasing dose of radiation and decreased after 90 min or repair incubation. Concomitant with these changes, the affinity of histones to DNA in chromatin reduced immediately after irradiation. Normal affinity was reestablished after post-irradiation repair incubation. The results indicate that induction of poly-ADP-ribosylation of histone proteins by radiation and by manipulations to prepare single cell suspension involved different cellular components.