No abstract

The annexins are family of calcium-regulated phospholipid-binding proteins with diverse roles in cell biology. Individual annexins have been implicated in tumour development and progression, and in this investigation a range of annexins have been studied in colorectal cancer. Annexins A1, A2, A4 and A11 were identified by comparative proteomic analysis to be overexpressed in colorectal cancer. Annexins A1, A2, A4 and A11 were further studied by immunohistochemistry with a colorectal cancer tissue microarray containing primary and metastatic colorectal cancer and also normal colon. There was significant increase in expression in annexins A1 (P ¼ 0.01), A2 (Po0.001), A4 (Po0.001) and A11 (Po0.001) in primary tumours compared with normal colon. There was increasing expression of annexins A2 (P ¼ 0.001), A4 (P ¼ 0.03) and A11 (P ¼ 0.006) with increasing tumour stage. An annexin expression profile was identified by k-means cluster analysis, and the annexin profile was associated with tumour stage (P ¼ 0.01) and also patient survival. Patients in annexin cluster group 1 (low annexin expression) had a better survival (log rank ¼ 5.33, P ¼ 0.02) than patients in cluster group 2 (high annexins A4 and A11 expression). In conclusion, this study has shown that individual annexins are present in colorectal cancer, specific annexins are overexpressed in colorectal cancer and the annexin expression profile is associated with survival.

The Wilms' tumor suppressor protein WT1 is a transcriptional regulator that plays a key role in the development of the kidneys. The transcriptional activation domain of WT1 is subject to regulation by a suppression region within the N terminus of WT1. Using a functional assay, we provide direct evidence that this requires a transcriptional cosuppressor, which we identify as brain acid soluble protein 1 (BASP1). WT1 and BASP1 associate within the nuclei of cells that naturally express both proteins. BASP1 can confer WT1 cosuppressor activity in transfection assays, and elimination of endogenous BASP1 expression augments transcriptional activation by WT1. BASP1 is present in the developing nephron structures of the embryonic kidney and, coincident with that of WT1, its expression is restricted to the highly specialized podocyte cells of the adult kidney. Taken together, our results show that BASP1 is a WT1-associated factor that can regulate WT1 transcriptional activity.Wilms' tumor, a pediatric malignancy of the kidneys, is the most common solid childhood tumor (reviewed in references 4, 7, 20, 30, and 33). The isolation of genes associated with Wilms' tumor led to the identification of a zinc finger protein, WT1. Subsequently, WT1 was shown to be a transcriptional regulator with putative target genes including those for growth factors and regulators of cell division (5,6,18,21). Approximately 15% of sporadic Wilms' tumors have been found to contain mutations in WT1, while others show aberrant WT1 expression (33).WT1 knockout mice (homozygous null) do not survive gestation, displaying absence or incorrect development of the kidney, gonads, spleen, heart, diaphragm, and retinal ganglia (12,14,35). These findings confirm a major role for WT1 in the formation of the genitourinary system and also a wider role in the development of other tissues.Alternative splicing, RNA editing, and an alternative translation start codon combine to produce a plethora of WT1 isoforms (reviewed in reference 33). One alternative splice inserts three amino acids (KTS) between zinc fingers three and four, resulting in a form of WT1 that associates with RNA processing factors and localizes to regions of RNA processing in the nucleus (17). Thus, the ϩKTS and ϪKTS isoforms of WT1 have been proposed to function in RNA processing and transcription, respectively. These isoforms have both overlapping and distinct roles during development (9, 10). Interestingly, the ϩKTS isoform of WT1 plays the dominant role in the development of the gonad, while the ϪKTS isoform has a more extensive function in kidney formation.The other alternative splice inserts 17 amino acids N terminal to the WT1 zinc fingers and has been shown to have effects on both cell division and cell survival (15,31,32). Specific elimination of this isoform of WT1 in mice does not result in any obvious defects in genitourinary development, suggesting that it may be required specifically for a tumor suppressor role or that it performs a subtle function (28).Several studies have shown ...

Heterogeneous ribonucleoprotein K (hnRNP K) is a member of the hnRNP family which has several different cellular roles including transcription, mRNA shuttling, RNA editing and translation. Several reports implicate hnRNP K having a role in tumorigenesis, for instance hnRNP K increases transcription of the oncogene c-myc and hnRNP K expression is regulated by the p53/MDM 2 pathway. In this study comparing normal colon to colorectal cancer by proteomics, hnRNP K was identified as being overexpressed in this type of cancer. Immunohistochemistry with a monoclonal antibody to hnRNP K (which we developed) on colorectal cancer tissue microarray, confirmed that hnRNP K was overexpressed in colorectal cancer (Po0.001) and also showed that hnRNP K had an aberrant subcellular localisation in cancer cells. In normal colon hnRNP K was exclusively nuclear whereas in colorectal cancer the protein localised both in the cytoplasm and the nucleus. There were significant increases in both nuclear (P ¼ 0.007) and cytoplasmic (P ¼ 0.001) expression of hnRNP K in Dukes C tumours compared with early stage tumours. In Dukes C patient's good survival was associated with increased hnRNP K nuclear expression (P ¼ 0.0093). To elaborate on the recent observation that hnRNP K is regulated by p53, the expression profiles of these two proteins were also analysed. There was no correlation between hnRNP K and p53 expression, however, patients who presented tumours that were positive for hnRNP K and p53 had a poorer survival outcome (P ¼ 0.045).

Specialized transporter proteins that are the products of two closely related genes, UT-A ( Slc14a2) and UT-B ( Slc14a1), modulate the movement of urea across cell membranes. The purpose of this study was to characterize the mouse variants of two major products of the UT-A gene, UT-A1 and UT-A2. Screening a mouse kidney inner medulla cDNA library yielded 4,047- and 2,876-bp cDNAs, the mouse homologues of UT-A1 and UT-A2. Northern blot analysis showed high levels of UT-A mRNAs in kidney medulla. UT-A transcripts were also present in testes, heart, brain, and liver. Immunoblots with an antiserum raised to the 19 COOH-terminal amino acids of rat UT-A1 (L194) identified immunoreactive proteins in kidney, testes, heart, brain, and liver and showed a complex pattern of differential expression. Relative to other tissues, kidney and brain had the highest levels of UT-A protein expression. In kidney sections, immunostaining with L194 revealed immunoreactive proteins in type 1 (short) and type 3 (long) thin descending limbs of the loop of Henle and in the middle and terminal inner medullary collecting ducts. Expression in Xenopus laevis oocytes showed that, characteristic of UT-A family members, the cDNAs encoded phloretin-inhibitable urea transporters. Acute application of PKA agonists (cAMP/forskolin/IBMX) caused a significant increase in UT-A1- and UT-A3-, but not UT-A2-mediated, urea transport.