Quantitative and mechanism-based information on differences in transfection efficiency between viral and non-viral vectors would be highly useful for improving the effectiveness of non-viral vectors. A previous quantitative comparison of intracellular trafficking between adenovirus and LipofectAMINE PLUS (LFN) revealed that the three orders of magnitude lower transfection efficiency of LFN was dominantly rate limited by the post-nuclear delivery process. In the present study, the contribution of transcription and translation processes to the overall differences in the transgene expression efficiency of nucleus-delivered DNA was independently evaluated by quantifying mRNA. As a result, transcription efficiency (Etranscript) of LFN, denoted as transgene expression divided by the amount of nuclear pDNA was about 16 times less than that for adenovirus. Furthermore, translation efficiency (Etranslate), denoted as transfection activity divided by mRNA expression was approximately 460 times less in LFN. Imaging of the decondensed form of DNA by in situ hybridization revealed that poor decondensation efficiency of LFN is involved in the inferior Etranscript. Moreover, the inferior translation efficiency (Etranslate) of LFN was mainly due to electrostatic interactions between LFN and mRNA. Collectively, an improvement in nuclear decondensation and the diminution of the interaction between vector and mRNA is essential for the development of new generations of non-viral vectors.

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Gene mutations were found in acute myeloid leukemia (AML) and their importance has been noted. To clarify the importance and stability of mutations, we examined gene mutations in paired samples at diagnosis and relapse of 34 adult AML patients. Five acquired gene mutations were detected at relapse. Of the 45 gene mutations at diagnosis, 11 of them were lost at relapse. The acquired mutations at relapse were all class I mutations as Fms-like tyrosine kinase 3 (FLT3) and rat sarcoma viral oncogene homolog (RAS) mutations. The disappeared mutations at relapse were 3 of 11 internal tandem duplications of FLT3 (FLT3-ITD) (27.3%), 3 of 3 FLT3 tyrosine kinase domain (FLT3-TKD) (100%), 3 of 13 Nucleophosmin 1 (23.1%) and 2 of 5 CCAAT/enhancer-binding protein-α (40%) mutations. However, epigenetics-modifying gene (DNMT3a, TET2 and IDH1/2) mutations had no change between diagnosis and relapse samples, and may become minimal residual disease marker. The frequency of FLT3-ITD at relapse in patients with DNMT3a mutation at diagnosis is significantly higher than those in patients without them (P=0.001). Moreover, the high frequency of FLT3-ITD at relapse is also seen in AML cases that initially present with any epigenetics-modifying gene mutations (P<0.001). Our results indicate that epigenetics-modifying gene mutations may cause genetic instability and induce FLT3-ITD, leading to resistance to therapy and relapse.

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Estrogens produced as a result of intratumoral aromatization has been recently shown to play important roles in proliferation of human non-small cell lung carcinomas (NSCLC), but the details have remained largely unknown. Therefore, in this study, we evaluated the possible roles of intratumoral aromatase in NSCLCs as follows: (a) evaluation of intratumoral localization of aromatase mRNA/protein in six lung adenocarcinoma cases using laser capture microdissection combined with quantitative reverse transcriptase-PCR and immunohistochemistry; (b) examination of the possible effects of isolated stromal cells from lung carcinoma tissues on aromatase mRNA transcript expression in lung carcinoma cell lines (A549 and LK87) through a coculture system; and (c) screening of cytokines derived from stromal LK001S and LK002S cells using cytokine antibody arrays and subsequent evaluation of effects of these cytokines on aromatase expression in A549 and LK87. Both aromatase mRNA and protein were mainly detected in intratumoral carcinoma cells but not in stromal cells. Aromatase expression of A549 and LK87 was upregulated in the presence of LK001S or LK002S cells. Several cytokines such as interleukin-6 (IL-6), oncostatin M, and tumor necrosis factor-α, all known as inducible factors of aromatase gene, were detected in conditioned media of LK001S and LK002S cells. Treatment of both oncostatin M and IL-6 induced aromatase gene expression in A549 an LK87, respectively. These results all indicated that intratumoral microenvironments, especially carcinoma-stromal cell interactions, play a pivotal role in the regulation of intratumoral estrogen synthesis through aromatase expression in human lung adenocarcinomas. Cancer Res; 70(16); 6659-69. ©2010 AACR.