Establishment of a doxycycline-regulated cell line with inducible, doubly-stable expression of the wild-type p53 gene from p53-deleted hepatocellular carcinoma cells

p53 is important in the development of hepatocellular carcinoma (HCC) and in therapeutic approaches, but the mechanism whereby it inhibits HCC growth is still unclear. The aim of the present study was to establish a HCC cell system in which p53 levels can be regulated. Full-length wild-type p53 cDNA obtained by PCR was cloned into a retroviral response vector controlled by the tetracycline responsive element (RevTRE-p53). The regulatory vectors RevTet-Off and RevTRE-p53 were transfected into a packaging cell line, PT67. Hep3B cells in which the p53 gene was deleted were infected with RevTet-Off viral particles from the PT67. Three G418-resistant cell clones with high luciferase expression and low background were infected with RevTRE-p53. By screening dozens of RevTRE-p53-infected clones with hygromycin we identified the one with the highest expression of p53 and the lowest background after doxycycline treatment. The results showed that p53 expression in this cell clone could be simply turned on or off by removing or adding doxycycline. Furthermore, it was found that the level of p53 protein was negatively and sensitively related to the doxycycline concentration. In conclusion, we have established a HCC cell line in which p53 expression can be switched on or off and regulated in a dose- and time-dependent manner

Screening for eukaryotic signal transduction and Mycobacterium isocitrate lyase inhibitor from actinomycetes and fungi of dipterocarp rain forests at Imabak Valey, Sabah, Malaysia

A diversity of actinomycetes and fungi was isolated from various sites during the Imbak Valley Scientific Expedition 2000. A total of 144 soil samples were collected under trees that have been identified to species or genus level. Imbak Valley is a lowland dipterocarp forest, which is interestingly dominated by Dryobalanops beccarii. Isolation of Streptomyces and non-Streptomyces actinomycetes on HV medium and other specific isolation media for non-Streptomyces yielded 203 isolates from 89 soil samples. Morphological characterisation of the isolated actinomycetes was carried out based on aerial mycelium colour, substrate mycelium colour and diffusible pigment production on oatmeal medium. Nine strains of fungi were isolated from the six soil samples plated on PDA medium. All actinomycetes isolates were grown under aerobic condition in liquid culture and extracted with acetone, and used for screening against proteins involved eukaryotic signal transduction. Yeast MAPK kinase and MAP kinase phosphatase were some of the targeted proteins used in this research. MKK1P386 and MKK1P386-MSG5 mutant yeasts were used to screen for these inhibitors, as these yeast kinase and phosphatase have homologous proteins in the MAP kinase signal transduction pathway in human. No inhibitors in the extracts were found in these screenings. Type 1 protein serine/ threonine phosphatase (GLC7) in yeast was used to screen inhibitors against PP1 inhibitors and no inhibitor was found. None of the fungal extracts showed any inhibitory activities in all the screening systems. No Ras/Raf inhibitor was found in the in vivo Ras/Raf interaction with the yeast two-hybrid screening system, which used to screen for inhibitor against Ras/ Raf protein interaction inhibitor. There were 11 actinomycetes extracts that showed toxicity against yeast strain LZ (transformant of Ras/ Raf). H7667, a Streptomycete toxic to yeast is further screened for inhibitors of the GSK3-beta pathway. H7763, a Streptomyces species that showed positive in the primary screen for inhibitor of isocitrate lyase (ICL) which is not itaconic acid (known ICL inhibitor). H7240 showed the strongest susceptibility towards the resin in which the concentration of 5g/l of resin is sufficient to produce growth inhibition of the bacteria

The telomere maintenance mechanism spectrum and its dynamics in gliomas

Background : The activation of the telomere maintenance mechanism (TMM) is one of the critical drivers of cancer cell immortality. In gliomas, TERT expression and TERT promoter mutation are considered to reliably indicate telomerase activation, while ATRX mutation and/or loss indicates an alternative lengthening of telomeres (ALT). However, these relationships have not been extensively validated in tumor tissues. Methods : Telomerase repeated amplification protocol (TRAP) and C-circle assays were used to profile and characterize the TMM cross-sectionally (n = 412) and temporally (n = 133) across glioma samples. WES, RNA-seq, and NanoString analyses were performed to identify and validate the genetic characteristics of the TMM groups. Results : We show through the direct measurement of telomerase activity and ALT in a large set of glioma samples that the TMM in glioma cannot be defined solely by the combination of telomerase activity and ALT, regardless of TERT expression, TERT promoter mutation, and ATRX loss. Moreover, we observed that a considerable proportion of gliomas lacked both telomerase activity and ALT. This telomerase activation-negative and ALT negative group exhibited evidence of slow growth potential. By analyzing a set of longitudinal samples from a separate cohort of glioma patients, we discovered that the TMM is not fixed and can change with glioma progression. Conclusions : This study suggests that the TMM is dynamic and reflects the plasticity and oncogenicity of tumor cells. Direct measurement of telomerase enzyme activity and evidence of ALT should be considered when defining TMM. An accurate understanding of the TMM in glioma is expected to provide important information for establishing cancer management strategies.This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF), funded by the Ministry of Science & ICT (NRF-2018M3A9H3021707), and the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI21C0239)

Maritime Interdiction Operations in Logistically Barren Environments

Includes supplementary materialThis report contains analysis that shows that existing technology exists to improve Maritime Interdiction Operations (MIO) by approximately 30%. Furthermore, analysis contained herein will aid MIO planning for future operations. Since MIOs are an inherently dangerous, but necessary activity with far reaching implications to theater political and economic dynamics, this improvement is of great interest. MIO is a Naval solution to the problems of smuggling weapons, explosives, people and narcotics. MIO, when employed correctly has the potential to save lives and limit economic/political damage.N

The telomere maintenance mechanism spectrum and its dynamics in gliomas

Background The activation of the telomere maintenance mechanism (TMM) is one of the critical drivers of cancer cell immortality. In gliomas, TERT expression and TERT promoter mutation are considered to reliably indicate telomerase activation, while ATRX mutation and/or loss indicates an alternative lengthening of telomeres (ALT). However, these relationships have not been extensively validated in tumor tissues. Methods Telomerase repeated amplification protocol (TRAP) and C-circle assays were used to profile and characterize the TMM cross-sectionally (n = 412) and temporally (n = 133) across glioma samples. WES, RNA-seq, and NanoString analyses were performed to identify and validate the genetic characteristics of the TMM groups. Results We show through the direct measurement of telomerase activity and ALT in a large set of glioma samples that the TMM in glioma cannot be defined solely by the combination of telomerase activity and ALT, regardless of TERT expression, TERT promoter mutation, and ATRX loss. Moreover, we observed that a considerable proportion of gliomas lacked both telomerase activity and ALT. This telomerase activation-negative and ALT negative group exhibited evidence of slow growth potential. By analyzing a set of longitudinal samples from a separate cohort of glioma patients, we discovered that the TMM is not fixed and can change with glioma progression. Conclusions This study suggests that the TMM is dynamic and reflects the plasticity and oncogenicity of tumor cells. Direct measurement of telomerase enzyme activity and evidence of ALT should be considered when defining TMM. An accurate understanding of the TMM in glioma is expected to provide important information for establishing cancer management strategies.N