Colorectal cancer (CRC) is the second most prevalent cause of cancer-related deaths in the Western world. 5-Fluorouracil (5-FU) is a standard chemotherapeutic drug to treat CRC. However, the response rate is less than 20% and patients who have responded to 5-FU may become resistant. Therefore there is an urgent need to examine the 5-FU response proteins so that patients with no response to 5-FU can change to other treatment strategies promptly. In this study, the proteomic expression profile in a CRC cell line SW480 before and after 5-FU treatment was examined using 2-dimensional electrophoresis technology. Fourteen proteins with differential expression were identified using mass spectrometry and 7 of them were validated using immunocytochemical (ICC) staining. Protein identification indicated that cyclophilin A, cytokeratin 19 (CK19), cytokeratin 8 (CK8), ras-related nuclear protein, heat shock protein 27 (hsp27) and peroxiredoxin 6 (Prx 6) were upregulated whereas heat shock protein 60 (hsp60), cytokeratin 18 (CK18), cytokeratin 9 (CK9), carbamoylphosphate synthetase I, α-enolase, heat shock protein 70 (hsp70), nm23 and ß-actin were down-regulated. Seven of the 14 proteins detected were validated by ICC staining, which showed that the expression of hsp27, Prx 6 and hsp70 correlated with that from proteomics profiling. Our results suggest that hsp27, Prx 6 and hsp70 are potential 5-FU response proteins and they may represent potential targets for further evaluation in other 5-FU-sensitive and-resistant CRC cell lines.
The rapid emergence of molecular diagnostic platforms has revolutionized the diagnostic approaches in hematology laboratory. Fluorescence in-situ hybridization, polymerase chain reaction and DNA sequencing are common techniques used in routine clinical laboratories for the diagnosis of hematological diseases. Different molecular techniques are indicated in different situations. This paper describes the utility of common molecular techniques. Fluorescence in-situ hybridization is specific for detection of chromosomal abnormalities using fluorescent labeled targeting probe. Polymerase chain reaction amplifies target DNA and reverse transcription polymerase chain reaction amplifies target RNA for the analysis of gene and its expression level. Real-time polymerase chain reaction is highly sensitive for detection of minimal residual disease in hemic malignancies. Gap-polymerase chain reaction is often employed for diagnosis of large deletions such as in alpha thalassemia. Allele-specific polymerase chain reaction is commonly used for single nucleotide polymorphism detection which is common in beta thalassemia, myeloproliferative neoplasm and acute leukemia. Inverse shifting-polymerase chain reaction can be employed for the detection of large genetic rearrangements such as those seen in hemophilia A. For genetically complex diseases such as hemophilia A, which involves a great variety of mutations in large genes, high resolution melting analysis can be used to scan for point mutations. Any suspected mutations are confirmed using post-PCR technologies, such as DNA sequencing. Although conventional diagnostic methods are able to provide a basic analysis in most cases, molecular technologies generate valuable genetic information that can refine diagnosis, better predict prognosis and facilitate disease monitoring. Fluorescence in situ hybridization (FISH)FISH was developed in early 80s and became one of the most sensitive assays for localization of specific nucleic acid sequences and detection of numerical chromosome abnormalities, structural chromosomal rearrangements and cryptic abnormalities [5]. Bone marrow aspirate and peripheral blood smears can be used for FISH analysis [2,4]. FISH makes use of fluorescent probes which hybridize only to complementary sequences and the resulting signals are examined under a fluorescent microscope. The utility of specific FISH J o ur nal o f M e d ic al Dia g n o s ti c Method s
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