Morphological revertants have been isolated from one line of adenovirus type 12-transformed hamster cells. This line, T637, is oncogenic in hamsters and contains multiple copies of the virus genome ...per cell. Different parts of the virus genome are represented in non-stoichiometric amounts and the virus DNA persists in the cells in an integrated form. The pattern of integrated virus genomes has been determined by the blotting technique. In the T637 line, morphological revertants arise spontaneously at relatively high frequency. Two of these revertants have been cloned. In contrast to the T637 line, the revertants F10 and G12 exhibit fibroblastic morphology. The patterns of integrated virus genomes in the revertants differs markedly from that of the T637 line; one of the revertant cell lines, F10, appears to have lost all virus DNA sequences. The morphological revertants continue to express the oncogenic phenotype, although the time required to produce tumours in animals appears to be prolonged compared to the parental BHK21 and the T637 cell lines. A number of biological parameters of the revertant lines have also been investigated.
In KB cells productively infected with adenovirus type 2, alkali-stable >100S and 40-100S viral DNAs are synthesized starting 2-4 hr postinfection, i.e., before unit length (34 S) viral DNA is made. ...The amount of >100S and 40-100S viral DNA increases when 34S viral DNA synthesis begins, and at 16-18 hr postinfection, the 40-100S viral DNA represents 5-20% of the total intracellular viral DNA. The 40-100S viral DNA is synthesized throughout infection. Part of the 40-100S DNA synthesized 5-8 hr postinfection has a density in alkaline CsCl gradients intermediate between those of viral and cellular DNAs. This finding indicates that newly synthesized viral DNA is covalently linked to cellular DNA. Viral sequences can be excised from the cellular DNA of infected cells with the EcoRI restriction endonuclease. Fragments of viral DNA are detected in polyacrylamide-agarose gels by DNA· DNA hybridization, and these fragments correspond in size to most of the known EcoRI fragments of adenovirus 2 DNA. Viral DNA sequences in size-classes between the EcoRI-A and -C fragments are also found and probably represent viral DNA linked to cellular sequences.
The physical state of the viral genome in four lines of hamster cells transformed by adenovirus type 12 (Ad12) has been investigated. The four lines of transformed cells originated from hamster cells ...after infection with Ad12 at multiplicities ranging from 5-350 plaque-forming units per cell. The DNA from transformed cells has been restricted with the Sal I endonuclease from Streptomyces albus which cleaves adenovirus DNA more frequently than DNA from adenovirus-transformed hamster cells. Thus after cleavage by the Sal I enzyme, it is possible to separate free adenovirus DNA sequences from these which are covalently linked to cellular DNA in transformed hamster cells. The results of sequential hybridization experiments in which the Sal I-treated DNA from transformed cells is first annealed to Ad12 DNA on filters, then eluted, and finally hybridized to hamster cell DNA, support the model of Ad12 DNA integrated in multiple fragments into the host genome. Further experiments will be required to characterize the host sequences adjacent to adenovirus DNA and to compare these sequences in different lines of Ad12 transformed cells.
Cisplatin is an extensively used chemotherapeutic drug for lung cancer, but the development of resistance decreases its effectiveness in the treatments of non-small cell lung cancer (NSCLC). In this ...study, we examined the effects of metformin, a widely used antidiabetic drug, on cisplatin radiosensitization in NSCLC cell lines. Human NSCLC cell lines, A549 (cisplatin-resistant) and H460 (cisplatin-sensitive), were treated with metformin, cisplatin or a combination of both drugs before ionizing radiation. Cell proliferation, clonogenic assays, western blotting, cisplatin-DNA adduct formation and immunocytochemistry were used to characterize the treatments effects. Metformin increased the radiosensitivity of NSCLC cells. Metformin showed additive and over-additive effects in combination with cisplatin and the radiation response in the clonogenic assay in H460 and A549 cell lines (p = 0.018 for the interaction effect between cisplatin and metformin), respectively. At the molecular level, metformin led to a significant increase in cisplatin-DNA adduct formation compared with cisplatin alone (p < 0.01, ANOVA-F test). This was accompanied by a decreased expression of the excision repair cross-complementation 1 expression (ERCC1), a key enzyme in nucleotide excision repair pathway. Furthermore, compared with each treatment alone metformin in combination with cisplatin yielded the lowest level of radiation-induced Rad51 foci, an essential protein of homologous recombination repair. Ionizing radiation-induced γ-H2AX and 53BP1 foci persisted longer in both cell lines in the presence of metformin. Pharmacological inhibition of AMP-activated protein kinase (AMPK) demonstrated that metformin enhances the radiosensitizing effect of cisplatin through an AMPK-dependent pathway only in H460 but not in A549 cells. Our results suggest that metformin can enhance the effect of combined cisplatin and radiotherapy in NSCLC and can sensitize these cells to radiation that are not sensitized by cisplatin alone.
Coenzyme Q10 is an essential cofactor in the electron transport chain and serves as an important antioxidant in both mitochondria and lipid membranes. CoQ10 is also an obligatory cofactor for the ...function of uncoupling proteins. Furthermore, dietary supplementation affecting CoQ10 levels has been shown in a number of organisms to cause multiple phenotypic effects. However, the molecular mechanisms to explain pleiotrophic effects of CoQ10 are not clear yet and it is likely that CoQ10 targets the expression of multiple genes. We therefore utilized gene expression profiling based on human oligonucleotide sequences to examine the expression in the human intestinal cell line CaCo-2 in relation to CoQ10 treatment. CoQ10 caused an increased expression of 694 genes at threshold-factor of 2.0 or more. Only one gene was down-regulated 1.5-2-fold. Real-time RT-PCR confirmed the differential expression for seven selected target genes. The identified genes encode proteins involved in cell signalling (n = 79), intermediary metabolism (n = 58), transport (n = 47), transcription control (n = 32), disease mutation (n = 24), phosphorylation (n = 19), embryonal development (n = 13) and binding (n = 9). In conclusion, these findings indicate a prominent role of CoQ10 as a potent gene regulator. The presently identified comprehensive list of genes regulated by CoQ10 may be used for further studies to identify the molecular mechanism of CoQ10 on gene expression.
Keratin 8 (KRT8) is one of the major intermediate filament proteins expressed in single-layered epithelia of the gastrointestinal tract. Transgenic mice over-expressing human KRT8 display pancreatic ...mononuclear infiltration, interstitial fibrosis and dysplasia of acinar cells resulting in exocrine pancreatic insufficiency. These experimental data are in accordance with a recent report describing an association between KRT8 variations and chronic pancreatitis. This prompted us to investigate KRT8 polymorphisms in patients with pancreatic disorders. The KRT8 Y54H and G62C polymorphisms were assessed in a cohort of patients with acute and chronic pancreatitis of various aetiologies or pancreatic cancer originating from Austria (n=16), the Czech Republic (n=90), Germany (n=1698), Great Britain (n=36), India (n=60), Italy (n=143), the Netherlands (n=128), Romania (n=3), Spain (n=133), and Switzerland (n=129). We also studied 4,234 control subjects from these countries and 1,492 control subjects originating from Benin, Cameroon, Ethiopia, Ecuador, and Turkey. Polymorphisms were analysed by melting curve analysis with fluorescence resonance energy transfer probes. The frequency of G62C did not differ between patients with acute or chronic pancreatitis, pancreatic adenocarcinoma and control individuals. The frequency of G62C varied in European populations from 0.4 to 3.8%, showing a northwest to southeast decline. The Y54H alteration was not detected in any of the 2,436 patients. Only 3/4,580 (0.07%) European, Turkish and Indian control subjects were heterozygous for Y54H in contrast to 34/951 (3.6%) control subjects of African descent. Our data suggest that the KRT8 alterations, Y54H and G62C, do not predispose patients to the development of pancreatitis or pancreatic cancer.
Coenzyme Q
10 is an essential cofactor in the electron transport chain and serves as an important antioxidant in both mitochondria and lipid membranes. CoQ
10 is also an obligatory cofactor for the ...function of uncoupling proteins. Furthermore, dietary supplementation affecting CoQ
10 levels has been shown in a number of organisms to cause multiple phenotypic effects. However, the molecular mechanisms to explain pleiotrophic effects of CoQ
10 are not clear yet and it is likely that CoQ
10 targets the expression of multiple genes. We therefore utilized gene expression profiling based on human oligonucleotide sequences to examine the expression in the human intestinal cell line CaCo-2 in relation to CoQ
10 treatment. CoQ
10 caused an increased expression of 694 genes at threshold-factor of 2.0 or more. Only one gene was down-regulated 1.5–2-fold. Real-time RT-PCR confirmed the differential expression for seven selected target genes. The identified genes encode proteins involved in cell signalling (
n
=
79), intermediary metabolism (
n
=
58), transport (
n
=
47), transcription control (
n
=
32), disease mutation (
n
=
24), phosphorylation (
n
=
19), embryonal development (
n
=
13) and binding (
n
=
9). In conclusion, these findings indicate a prominent role of CoQ
10 as a potent gene regulator. The presently identified comprehensive list of genes regulated by CoQ
10 may be used for further studies to identify the molecular mechanism of CoQ
10 on gene expression.
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