The PFA-AMeX method achieves a good balance between the morphology of tissues and the quality of RNA content in DNA microarray analysis with laser-capture microdissection samples.

Recently, large-scale gene expression profiling is often performed using RNA extracted from unfixed frozen or formalin-fixed paraffin embedded (FFPE) samples. However, both types of samples have drawbacks in terms of the morphological preservation and RNA quality. In the present study, we investigated 30 human prostate tissues using the PFA-AMeX method (fixation using paraformaldehyde (PFA) followed by embedding in paraffin by AMeX) with a DNA microarray combined with laser-capture microdissection. Morphologically, in contrast to the case of atypical adenomatous hyperplasia, loss of basal cells in prostate adenocarcinomas was as obvious in PFA-AMeX samples as in FFPE samples. As for quality, the loss of rRNA peaks 18S and 28S on the capillary electropherograms from both FFPE and PFA-AMeX samples showed that the RNA was degraded equally during processing. However, qRT-PCR with 3' and 5' primer sets designed against human beta-actin revealed that, although RNA degradation occurred in both methods, it occurred more mildly in the PFA-AMeX samples. In conclusion, the PFA-AMeX method is good with respect to morphology and RNA quality, which makes it a promising tool for DNA microarrays combined with laser-capture microdissection, and if the appropriate RNA quality criteria are used, the capture of credible GeneChip data is well over 80% efficient, at least in human prostate specimens.

Involvement of glypican-3 in the recruitment of M2-polarized tumor-associated macrophages in hepatocellular carcinoma.

Previously, we demonstrated that membrane expression of glypican-3 (GPC3) stimulates the recruitment of macrophages into human hepatocellular carcinoma (HCC) tissues. However, functional polarization of the macrophages and the chemoattractant factors related to the recruitment has yet to be determined. In this study, to clarify the polarization (M1 or M2) of the macrophages and provide a clue as to the factors involved in the recruitment, we used xenograft models of SK-HEP-1 and SK03 cell lines with undetectable and high-level membrane expression of GPC3, respectively and analyzed the expression profiles of the relevant genes in both xenografts mainly using microarray techniques. Clustering analyses with mouse genome arrays revealed that the SK-HEP-1 and SK03 xenografts showed different expression profiles for M2 macrophage-related genes but not for M1 macrophage-related genes. Many of the M2 macrophage-related genes were upregulated in the SK03 xenografts compared to the SK-HEP-1 xenografts. Additionally, most of the macrophages infiltrating into the SK03 xenografts were positive for M2 macrophage-specific markers. Regarding the chemoattractant factors, the microarray and quantitative real-time PCR analyses revealed that, of the genes reportedly related to macrophage recruitment, CCL5, CCL3 and CSF1 were significantly upregulated in the SK03 xenograft. These findings suggest that the macrophages recruited into GPC3-overexpressing (with membrane expression) HCC are M2-polarized ones and, more specifically, M2 tumor-associated macrophages which are known to promote tumor progression and metastasis, and CCL5, CCL3 and CSF1 are possible candidate genes for the recruitment of macrophages.

Gene amplification and expression in lung cancer cells with acquired paclitaxel resistance.

A paclitaxel-resistant subline was generated from the non-small lung cancer cell line NCI-H460 by stepwise selection in paclitaxel from 0.032 to 250 nmol/L. The resulting subline, designated NCI-H460/PTX250, showed 792-fold resistance against paclitaxel compared with the parental cell line NCI-H460. The chemosensitivity analysis revealed the cross-resistance phenotype against various anticancer drugs including docetaxel, vinblastine, and doxorubicin, but not against camptotecin, cisplatin, and 5-fluorouracil. The addition of 5 mumol/L verapamil or reversin 121 reversed the resistance against paclitaxel, vinblastine, and doxorubicin. The gene expression profile, examined using oligonucleotide microarrays, demonstrated that the expression of 332 and 342 genes was significantly increased and decreased, respectively, in NCI-H460/PTX250 compared with NCI-H460. The most highly upregulated gene was MDR1/ABCB1 with a 1,092-fold increase. The overexpression was confirmed at the protein level by Western blot and flow cytometry analyses. The copy number profile, examined using microarray-based comparative genomic hybridization, revealed amplification of the q11.21 approximately q21.12 region on chromosome 7. In particular, the entire q21.12 region displayed 11- to 13-fold higher copy number in NCI-H460/PTX250 than in NCI-H460. Most of the genes within the region were highly expressed, and the increased expression of these genes could be explained by the amplification in the gene copy number. However, the increase in MDR1/ABCB1 expression greatly exceeded the genomic copy number increase of the gene, suggesting the existence of one or more additional factors, such as transcriptional enhancement or mRNA stabilization, associated with the elevated MDR1/ABCB1 expression. In conclusion, both chromosomal region-specific copy number amplification and gene-specific activation are probably involved in the overexpression of MDR1/ABCB1, resulting in acquisition of the drug resistance phenotype in NCI-H460/PTX250.