Alternative splicing of human cytokines and its role in carcinogenesis

Abstract

Expression genetics is becoming a new approach to the identification of cancer-related genes. In this point, cytokines seems to have an important role since it systemically regulates cells. They contribute to a wide range of symptoms in advanced cancers. Aberrant expressions in cytokines have been reported in some cancer types and stages. Alternative splicing is an essential biological process that generates multiple different transcripts from the same precursor mRNA. It is an important regulatory mechanism for high eukaryotic gene expression. Recently, inherited and acquired changes in pre-mRNA splicing have been also documented to play a significant role in human disease development and many cancer-associated genes are regulated by alternative splicing. Here, the author focused on the alternative splicing of human granulocyte-colony stimulating factor (hG-CSF) and reports that exon3 skipping of hG-CSF is more related to carcinogenesis. In part I, alternative splicing event of hG-CSF from different cell lines were investigated by RT-PCR, southern hybridization, northern hybridization and DNA chip hybridization. Among different splicing variants of hG-CSF, it was concluded that exon 3 skipping is more frequent event, in particular, in various cancer samples. An attempt to investigate the exon 3 skipping in cancer was firstly made with different cell lines by development of DNA chip which has a specific and sensitive DNA probes for differentiating each hG-CSF variant type. Clinical samples from tissues and blood were tested for analyzing exon 3 skipped hG-CSF (mG-CSF) expression frequencies. In Fisher’s Exact Test, there was statistically significant difference between cancer group and non-cancer group in tissues and non-cancer samples even though the frequency is lower than expected. However, there was no statistically significant difference between normal section and cancer section of cancer tissues, which means that exon3 skipping of hG-CSF is strongly associated with cancer and/or carcinogenesis. In part II, mG-CSF expression induced by carcinogen treatment and normal hG-CSF was discussed. Thioacetamide (TA) is a weak carcinogen specific for the liver carcinogenesis. Its short term exposure gives reversible premalignant states. It was examined that carcinogen TA treatment induced hG-CSF and mG-CSF expression. It is likely that mG-CSF and hG-CSF expression seem to be early response genes since they expresses within 2 to 4 hours after TA treatment. Recently, it was reported that hG-CSF expression correlates with increased tumor proliferation and vascularization in human meningiomas. Increased cytokine expression has been reported in many types of cancers. In particular, mG-CSF expression may be positively related to carcinogenesis in addition to the increased cytokine expression. mG-CSF expression may be also positively related to hG-CSF expression and carcinogenesis. We also found that hG-CSF induces mG-CSF expression which seems to be by p38 and JNK pathway. In part III, simple patterning of biomolecules by using photolithography was discussed. For the further application of these newly found cancer-associated genes and proteins, development of molecular cancer detection system is also useful and if it is combined with patterning methods, it can be used for various purposes since DNA chip is still limited to DNA based assay. The author reported the very simple immobilization methods for the further application for cell-protein-and DNA based assay. In this thesis, cells and DNAs were successfully immobilized and patterned with having its own activity, which opens new assay method applicable in various different fields such as biosensors for monitoring and diagnosis and/or cell engineering for constructing mimicking in vivo system to study the effects on cells. An attempt was also made to diagnose most common corneal dystrophies caused by mutations in the $TGF-\beta$ induced ($\beta igh3$) gene by utilizing the chip system, which was successfully applied to cancer diagnosis based on the alternative splicing of G-CSFs in previous chapters. The author successfully diagnosed most common dystrophies with chip system.

As more papers still describe the cancer mechanisms and effective cancer markers including this newly found mG-CSF, the question arises whether the above statement could be useful tool for cancer diagnosis. The function and diagnostic importance of this newly found mG-CSF expression are needed to be studied more in detail. However, like classical biomarkers, this newly found mG-CSF has been already shown to correlate with malignancy in cancer cell lines, tissues and even in blood. Now an effort is being made to identify the novel mG-CSF protein and its usefulness as a universal cancer marker. Undoubtly, the examination between these aberrant expression of cytokines including hG-CSF and cancer will prove to be an extremely useful for the identification of a new generation of biomarkers.