Application of RT-PCR in formalin-fixed and paraffin-embedded lung cancer tissues.

AIM: To analyze gene expression in formalin-fixed, paraffin-embedded lung cancer tissues using modified method. METHODS: Total RNA from frozen tissues was extracted using TRIZOL reagent. RNA was extracted from formalin-fixed, paraffin-embedded tissues by digestion with proteinase K before the acid-phenol:chloroform extraction and carrier precipitation. We modified this method by using a higher concentration of proteinase K and a longer digestion time, optimized to 16 hours. RT-PCR and real-time RT-PCR were used to check reproducibility and the concordance between frozen and paraffin-embedded samples. RESULTS: The results showed that the RNA extracted from the paraffin-embedded lung tissues had high quality with the most fragment length between 28S and 18S bands (about 1000 to 2000 bases). The housekeeping gene GUSB exhibited low variation of expression in frozen and paraffin-embedded lung tissues, whereas PGK1 had the lowest variation in lymphoma tissues. Furthermore, real-time PCR analysis of the expression of known prognostic genes in non-small cell lung carcinoma (NSCLC) demonstrated an extremely high correlation (r>0.880) between the paired frozen and formalin-fixed, paraffin-embedded specimens. CONCLUSION: This improved method of RNA extraction is suitable for real-time quantitative RT-PCR, and may be used for global gene expression profiling of paraffin-embedded tissues.

Different BAG-1 isoforms have distinct functions in modulating chemotherapeutic-induced apoptosis in breast cancer cells.

AIM: BAG-1 is a multifunctional anti-apoptotic gene with four isoforms, and different BAG-1 isoforms have different anti-apoptotic functions. In this study, we transfected BAG-1 isoforms into the human breast cancer cell lines Hs578T (ER negative) and MCF-7 (ER positive) to study their effect on apoptosis with or without estrogens. METHODS: The constructed recombinant expression vectors carrying individual BAG-1 isoforms was used to transfect human breast cancer cell lines Hs578T (ER negative) and MCF-7 (ER positive). After stable cell lines were made, a variety of apoptosis-inducing agents, including doxorubicin, docetaxel, and 5-FU, was used to treat these cell lines with or without estrogen to test the role of BAG-1. The mechanism by which BAG-1 affected the function of Bcl-2 was exploredby using the cycloheximide chase assay. RESULTS: The BAG-1 p50 and p46 isoforms significantly enhanced the resistance to apoptosis in both cell lines according to flow cytometry analysis. BAG-1 p33 and p29 failed to protect the transfected cells from apoptosis. The cell viability assay showed that only BAG-1 p50, but not p46, p33, or p29, increased estrogen-dependent function in ER-positive cell line MCF-7. Only BAG-1 p50 dramatically increased its anti-apoptotic ability in the presence of estrogen, while estrogen has very little effect on the anti-apoptotic ability of other BAG-1 isoforms. In the detection of the expression of K-ras, Hsp70, cytochrome c, Raf-1, ER-alpha, and Bcl-2 in MCF-7 cells by Western blot, only Bcl-2 protein expression was significantly increased in MCF-7 cells transfected with BAG-1 p50 and p46, respectively. Furthermore, the cycloheximide chase assay indicated that the degradation of Bcl-2 protein was extended in the BAG-1 p50 and p46 transfected MCF-7 cells. CONCLUSION: Distinct isoforms of BAG-1 have different anti-apoptotic functions in breast cancer cells, and that the BAG-1 p50 isoform can potentiate the role of estrogen in ER-positive breast cancer.