The identification of specific and reproducible biomarkers is critical for the early diagnosis of cancer, which has a profound effect the survival rate of patients. Comprehensive laboratory and clinical evidence needs to be collected to confirm the accuracy of the biomarkers prior to their clinical use. Heterogenous nuclear ribonucleoproteins (hnRNPs) A2 and B1 have been suggested as biomarkers for cancer since 1988 when hnRNP A2/B1 overexpression was first linked with the occurrence of lung cancer. Later studies established a correlation between the expression levels of these hnRNPs and other cancers, such as breast, pancreatic, and lymphatic tumours. In this study, the expression of hnRNPs A1, A2, A3, and B1 has been investigated in various cancer cell lines. hnRNPs A1 and A3, in addition to A2 and B1, were found to be overexpressed in some cancer types. However, the overexpression of none of the hnRNP A/B proteins was universal, and their upregulation may be limited to a few cell types, suggesting they may be effective biomarkers for a subset of cancers. The upregulation of hnRNP A/B proteins in tumours and cancer cell lines led to the hypothesis that they are involved in the uncontrolled cell growth in cancer. According to our Western blot analysis, expression of the hnRNP proteins, A1, A2, and B1, is dependent on the cell cycle whereas no significant change was detected for hnRNP A3, implying that the former three are needed during certain cell cycle stages. The results, together with the transcription factor analysis of the promoter regions of the HNRPA1, HNRPA2, and HNRPA3 genes, suggest that hnRNPs A1, A2, and A3 may have distinct regulatory machineries and cellular functions although they have high amino acid sequence identity. However, their mRNA levels were unchanged across the cell cycle, suggesting the cell-cycle-dependent expression of hnRNPs A1, A2, and B1 is modulated at the translational level. Previous studies showed higher expression of hnRNPs A1 and A2 in rapidly proliferating cells than in quiescent cells, suggesting a role of these proteins in cell proliferation. Though interruption of hnRNP A1 expression did not result in significant change in the viability of murine CB3 cells, simultaneous suppression of hnRNPs A1 and A2 caused apoptosis in a few cell lines. Consistent with this, suppression of hnRNP A1 or A3 expression in our study in Colo16 squamous cells using RNA interference did not affect cell proliferation, but simultaneous suppression of both caused slow cell proliferation. By contrast, reduction of the hnRNP A2 level alone slowed the proliferation of Colo16 cells. These results suggest that although hnRNPs A1, A2, and A3 share some roles in cell proliferation, each of them may have distinct tasks. This conclusion is supported by the data from the comparative analysis of the downstream targets of hnRNPs A1, A2, and A3, which has shown that these three proteins share a limited number of common downstream proteins. The observed impact on cell proliferation of suppressing hnRNP A2 subfamily proteins is in accord with our finding that the downstream targets of hnRNP A2 are overrepresented by genes involved in proliferation regulation, as shown in microarray and real-time PCR analysis. These include cyclin-dependent kinase (CDK) inhibitors, p21 and p27, and their regulatory proteins, such as Skp2 and Rpn10. Skp2 controls the ubiquitination of p21 and p27, and Rpn10 links them to the 26S proteasome, the complex that degrades these two CDK inhibitors. hnRNP A2 also regulates the transcription of securin and separin, which are essential for sister chromatid separation during late anaphase. In addition, hnRNP A2 can also influence cell proliferation through cell growth factors, including fibroblast, vascular endothelial, transforming, and insulin growth factors. Our gene array and real-time PCR analysis have shown that hnRNP A2 regulates the expression of these factors, their receptors, or associated proteins such as IGFBP7 and TGFBR2. The data presented in this thesis link the overexpression of hnRNP A/B proteins, in particular the A2/B1 subfamily, in cancer with their regulatory roles in cell division and cell proliferation. Our findings provide mechanistic evidence that these proteins may be a driving force for the uncontrolled cell growth in cancer, suggesting that some of hnRNP A/B proteins may be potential therapeutic targets for cancer. However, further studies are needed to obtain a global view of the roles of these proteins in cancer.

No description available.

The identification of specific and reproducible biomarkers is critical for the early diagnosis of cancer, which has a profound effect the survival rate of patients. Comprehensive laboratory and clinical evidence needs to be collected to confirm the accuracy of the biomarkers prior to their clinical use. Heterogenous nuclear ribonucleoproteins (hnRNPs) A2 and B1 have been suggested as biomarkers for cancer since 1988 when hnRNP A2/B1 overexpression was first linked with the occurrence of lung cancer. Later studies established a correlation between the expression levels of these hnRNPs and other cancers, such as breast, pancreatic, and lymphatic tumours. In this study, the expression of hnRNPs A1, A2, A3, and B1 has been investigated in various cancer cell lines. hnRNPs A1 and A3, in addition to A2 and B1, were found to be overexpressed in some cancer types. However, the overexpression of none of the hnRNP A/B proteins was universal, and their upregulation may be limited to a few cell types, suggesting they may be effective biomarkers for a subset of cancers. The upregulation of hnRNP A/B proteins in tumours and cancer cell lines led to the hypothesis that they are involved in the uncontrolled cell growth in cancer. According to our Western blot analysis, expression of the hnRNP proteins, A1, A2, and B1, is dependent on the cell cycle whereas no significant change was detected for hnRNP A3, implying that the former three are needed during certain cell cycle stages. The results, together with the transcription factor analysis of the promoter regions of the HNRPA1, HNRPA2, and HNRPA3 genes, suggest that hnRNPs A1, A2, and A3 may have distinct regulatory machineries and cellular functions although they have high amino acid sequence identity. However, their mRNA levels were unchanged across the cell cycle, suggesting the cell-cycle-dependent expression of hnRNPs A1, A2, and B1 is modulated at the translational level. Previous studies showed higher expression of hnRNPs A1 and A2 in rapidly proliferating cells than in quiescent cells, suggesting a role of these proteins in cell proliferation. Though interruption of hnRNP A1 expression did not result in significant change in the viability of murine CB3 cells, simultaneous suppression of hnRNPs A1 and A2 caused apoptosis in a few cell lines. Consistent with this, suppression of hnRNP A1 or A3 expression in our study in Colo16 squamous cells using RNA interference did not affect cell proliferation, but simultaneous suppression of both caused slow cell proliferation. By contrast, reduction of the hnRNP A2 level alone slowed the proliferation of Colo16 cells. These results suggest that although hnRNPs A1, A2, and A3 share some roles in cell proliferation, each of them may have distinct tasks. This conclusion is supported by the data from the comparative analysis of the downstream targets of hnRNPs A1, A2, and A3, which has shown that these three proteins share a limited number of common downstream proteins. The observed impact on cell proliferation of suppressing hnRNP A2 subfamily proteins is in accord with our finding that the downstream targets of hnRNP A2 are overrepresented by genes involved in proliferation regulation, as shown in microarray and real-time PCR analysis. These include cyclin-dependent kinase (CDK) inhibitors, p21 and p27, and their regulatory proteins, such as Skp2 and Rpn10. Skp2 controls the ubiquitination of p21 and p27, and Rpn10 links them to the 26S proteasome, the complex that degrades these two CDK inhibitors. hnRNP A2 also regulates the transcription of securin and separin, which are essential for sister chromatid separation during late anaphase. In addition, hnRNP A2 can also influence cell proliferation through cell growth factors, including fibroblast, vascular endothelial, transforming, and insulin growth factors. Our gene array and real-time PCR analysis have shown that hnRNP A2 regulates the expression of these factors, their receptors, or associated proteins such as IGFBP7 and TGFBR2. The data presented in this thesis link the overexpression of hnRNP A/B proteins, in particular the A2/B1 subfamily, in cancer with their regulatory roles in cell division and cell proliferation. Our findings provide mechanistic evidence that these proteins may be a driving force for the uncontrolled cell growth in cancer, suggesting that some of hnRNP A/B proteins may be potential therapeutic targets for cancer. However, further studies are needed to obtain a global view of the roles of these proteins in cancer.

The GTP hydrolases (GTPases) are a ubiquitous superfamily of proteins with diverse cellular roles. Members of the recently-discovered DRG subfamily have been implicated in human disease and may play a role in cell division, differentiation or death. While recent work in our laboratory has focused on the expression and subcellular localisation of DRG1 and DRG2 in the model plant Arabidopsis thaliana (Etheridge et al., 1999; Etheridge, 2002), our understanding of the role of these proteins in planta remains unclear. A yeast two-hybrid (Y2H) library screen using Arabidopsis DRG2 as bait identified 16 interacting proteins, 75% of which are predicted to reside in cellular compartments other than the cytoplasm. Since DRG2 has previously been localised to cytoplasmic vesicles, this observation suggests that DRG2 could be involved in the trafficking of these proteins to their correct cellular locations. A single DRG2 interactor, DRGIP4, was selected for detailed study and its interaction with DRG2 was confirmed in vivo and in vitro. Based on sequence similarity to Pseudomonas PCD/DCoH, DRGIP4 was renamed PDL1 (PCD/DCoH-like protein 1). Members of the widespread PCD/DCoH family are bifunctional proteins which possess catalytic and transcriptional coactivation functions in different cellular compartments and in different oligomeric states. PDL1 is encoded by a single-copy gene in Arabidopsis and shares remarkable secondary and tertiary structural conservation with members of the PCD/DCoH family and is also capable of homomeric associations. PDL1 contains an N-terminal chloroplast transit peptide (cTP) which is functional in planta and is important for interaction with DRG2, suggesting that these proteins interact in the cytoplasm prior to the import of PDL1 into the chloroplast. PDL1 lacks most of the residues important for PCD/DCoH enzymatic activity but may retain a transcriptional coactivation function in the chloroplast. PDL1 homologs in other plants also contain an N-terminal cTP and a PCD/DCoH domain. pdl1 mRNA is expressed at moderate to high levels in the aerial tissues but not in the roots. Accumulation of pdl1 transcripts is light-inducible, and the pdl1 promoter contains several cis-elements which may be responsible for light-responsive transcription. Expression of the intron-GUS reporter gene under the control of the pdl1 promoter generally correlates with mRNA accumulation but reveals tight spatial control of gene expression. Overexpression of pdl1 in transgenic plants does not result in an obvious phenotype. However, downregulation of pdl1 in transgenic plants results in a pale green leaf phenotype associated with a reduction in photosynthetic pigment content and chloroplast numbers per cell. Leaf internal architecture and chloroplast ultrastructure are unaffected in these plants. This phenotype is similar to the arc (accumulation and replication of chloroplasts) mutants and mutants in the chloroplast division and protein import machinery. PDL1 may therefore be involved in the process or regulation of chloroplast division in Arabidopsis. The pdl1 downregulation phenotype is also associated with pleiotropic effects on plant growth. A second PCD/DCoH-like protein, PDL2, was identified in the Arabidopsis genome sequence. Like PDL1, PDL2 shares limited primary sequence similarity to members of the PCD/DCoH family but retains characteristic secondary and tertiary structural features. PDL2 contains an N-terminal mitochondrial transit peptide (mTP) and is expressed at low levels in all plant tissues. PDL2 homologs in other plants also contain an N-terminal mTP and a PCD/DCoH domain. The significance of PDL2 for the function of PDL1 and DRG2 is not yet clear. Overexpression of drg1 in transgenic plants does not result in an obvious phenotype, while downregulation of drg1 in transgenic plants affects general aspects of plant growth which may be unrelated to DRG1 function. To investigate the role of DRGs in a different model system, homologs of drg1 and drg2 were isolated from Saccharomyces cerevisiae (baker’s yeast) and Schizosaccharomyces pombe (fission yeast). Knockout of either or both genes in transgenic yeast is non-lethal and results in sensitivity to agents which disrupt intra- and inter-molecular protein interactions. This phenotype is consistent with a role in protein trafficking. Overexpression of drg1 or drg2 in S. cerevisiae does not result in an obvious phenotype, while overexpression of either gene in S. pombe results in a slow growth phenotype. DRG1 and DRG2 are localised in the cytoplasm in S. pombe. The results presented here suggest that DRGs may be involved in the trafficking of proteins to different subcellular compartments. This research has provided a foundation for the detailed functional characterisation of plant and yeast DRGs and of the novel PDL family in Arabidopsis.

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