Previous studies of transformed rodent fibroblasts have suggested that specific isoforms of the actin-binding protein tropomyosin (TM) could function as suppressors of transformation, but an analysis of TM expression in patient tumor tissue is limited. The purpose of our study was to characterize expression of the different TM isoforms in human transitional cell carcinoma of the urinary bladder by immunohistochemistry and Western blot analysis. We found that TM1 and TM2 protein levels were markedly reduced and showed >60% reduction in 61% and 55% of tumor samples, respectively. TM5, which was expressed at very low levels in normal bladder mucosa, exhibited aberrant expression in 91% of tumor specimens. The Western blot findings were confirmed by immunohistochemical analysis in a number of tumors. We then investigated the mechanism underlying TM expression deregulation, in the T24 human bladder cancer cell line. We showed that levels of TM1, TM2 and TM3 are reduced in T24 cells, but significantly upregulated by inhibition of the mitogen-activated protein kinase-signaling pathway. In addition, inhibition of this pathway was accompanied by restoration of stress fibers. Overall, changes in TM expression levels seem to be an early event during bladder carcinogenesis. We conclude that alterations in TM isoform expression may provide further insight into malignant transformation in transitional cell carcinomas of the bladder and may be a useful target for early detection strategies. © 2004 Wiley-Liss, Inc. Key words: tropomyosin; cytoskeleton; bladder neoplasmsTransitional cell carcinoma (TCC) is the most common cancer type of the urinary bladder, representing approximately 90% of all cases. More than 57,000 people are diagnosed with TCC of the urinary bladder each year in the United States alone, and 12,500 of these patients are expected to die from the cancer. 1 Cytoscopy is the most valuable method to detect and monitor TCC, but it remains difficult to predict tumor progression, optimal therapy and finally clinical outcome. 2,3 There is a clear need for developing biomarkers for bladder cancer management.Alterations of the actin-based cytoskeleton are an established part of the neoplastic phenotype, and it is now demonstrated that such alteration is not a byproduct of cellular transformation but contributes to malignant transformation. 4,5 These alterations in actin remodeling are associated with downregulation of numerous actin-binding proteins and cell adhesion molecules, which are therefore candidate markers for early cancer detection and prognostic indication. 6 Tropomyosins (TMs) are a family of actin-binding proteins essential for the integrity of actin filaments. 7 Although TMs have been known to function in regulation of muscle contraction, the functional significance of the multiple TM isoforms present in non-muscle cells has remained largely unclear. Several studies have suggested that specific isoforms of TMs may possess tumor suppressor activity. Consistent with this hypothesis it was shown that (i) expr...

We have identi®ed a novel cDNA product designated transitional epithelial response gene (TERE1), which was localized to chromosome 1p36. The TERE1 transcript (1.5 and 3.5 kb) is present in most normal human tissues including urothelium, but was reduced or absent in the majority of muscle invasive TCC tumors (22 out of 29 cases). The open reading frame encodes a protein of 338 amino acids (MW 36.8 KD). This protein is 57% homologous to a Drosophila protein called heix. We have shown by Western blotting and immuno-histochemistry with a polyclonal antibody to a speci®c TERE1 peptide, reduced or absent staining in muscle invasive tumors. Transfection of a sense TERE1 construct resulted in an 80 ± 90% inhibition of cellular proliferation in two TCC cell lines and a lack of aneuploidy in the TERE1-transduced J82 cell line. These data suggest a potential role for this gene product in the progression of bladder cancer. Oncogene (2001) 20, 1042 ± 1051.

TERE1, a recently discovered gene/protein appears to play a role in bladder tumor growth regulation but to date does not have clear functional correlates. The objective of this study was to gain further insight into the function of the TERE1 protein by identifying potential protein to protein interactions with TERE1 and determining whether these interactions are associated with putative growth regulatory pathways and/or bladder tumor formation. Towards this aim, we have performed a bacterial two hybrid assay and isolated interacting clones, which then were sequenced and further examined by affinity chromatography and immunoprecipitation. From among several positive clones, we isolated a putative interacting plasmid containing the C-terminal portion of preapolipoprotein E starting from amino acid number 124 from the pBT-TERE1/pTarget-cDNA bacterial two hybrid system. The C-terminal portion of apoE interaction with the TERE1 was confirmed using ProBond columns by the expression of 6XHis recombinant and (35)S methionine/cysteine labeled proteins. We found that there was ubiquitous expression of the apoE transcript in normal bladder and in various grades and stages of transitional cell carcinoma (TCC) of the bladder. Likewise, we detected the apoE protein in both normal and malignant bladder tissues by Western blot. There was a significant decrease in the apoE protein in 12 of 16 muscle invasive TCCs of the bladder compared to normal bladder mucosa samples. Previous studies in rat fibroblasts have found that expression of apoE can decrease the phosphorylation of the growth factor-related p42/44 MAP kinase. A significant decrease in p44/p42 MAPK phophorylation was also apparent using a phosphorylation specific antibody in human 293 kidney cells upon transfection and expression of apoE. In conclusion, the results from this study suggest that the expression and regulation of the apoE pathway may yield clues toward understanding the function of TERE1.

Recently, we isolated a ubiquitously expressed gene designated TERE1, which has a significant effect on the growth regulation in bladder cancer. The TERE1 gene maps to chromosome 1p36.11-1p36.33 between the micro-satellite markers D1S2667 and D1S434, a chromosome locus that has been identified by loss of heterozygosity studies as a site of a putative tumor suppressor gene or genes for multiple tumor types including prostate carcinoma. The expression of the TERE1 transcript and protein was examined in a series of thirty microdissected prostate tumors by semi-quantitative RT/PCR and immunohistochemistry. There was a significant 61% decrease in the TERE1 transcript in prostate carcinoma (CaP) and a distinct loss of the TERE1 protein in metstatic prostate. Though a loss of heterozygosity at chromosome 1p36 was found in 25% of these prostate tumors, there appeared to be no TERE1 mutations present in these tumor samples. Induced TERE1 expression after transduction or transfection of TERE1 constructs into two prostate carcinoma (LNCaP and PC-3) cell lines significantly decreased proliferation up to 80% with a significant increase in the number of cells in G1. Serum factors but not DHT (dihydrotestosterone) appear to regulate the amount of TERE1 protein in the androgen responsive LNCaP cell line. Additionally, we have identified by microarray analysis various growth regulatory genes that are down-regulated or up-regulated in TERE1-transduced PC-3 cells. Altogether, these data suggest that TERE1 maybe significant in prostate cancer growth regulation and the down regulation or absence of TERE1 may be an important component of the phenotype of advanced disease.

In this investigation, we examined the role of the Axl proto-oncogene in renal cell carcinoma (RCC). Axl is a tyrosine kinase receptor implicated in myeloid leukogenesis, and has been found to be overexpressed in lung cancers and breast cancers. Axl has been described to act as a mitogenic factor along with its ligand Gas-6. Axl has also shown to have a role in apoptosis, cell adhesion, and chemotaxis. The differential expression of the Axl RNA transcript was examined in 20 pairs of matched normal kidney and clear cell RCC patient samples. We found that there was a significant increase in the steady-state levels of Axl mRNA in the RCC compared with the normal kidney pair (Student's paired t-test P < 0.001). There was also a significant increase in Axl expression overall in RCC compared to normal kidney (P < 0.03). Western blotting was utilized to determine Axl protein levels in six out of the 20 pairs of the normal/RCC matched pairs. Overall, the level of expression was not significantly different between the paired normal kidneys and kidney tumors, but the detected Axl protein appeared to be at slightly different molecular weights. Primers were constructed for the two known Axl variant, RT-PCR performed, but no differences were observed in the expression of each variant. Next, we performed a gene silencing experiment utilizing double-stranded RNA constructed to silence the Axl gene in the 293 transformed kidney cell line. There was a 50% decrease in Axl gene expression in the RNAi transfected over control cells. In addition, flow cytometry performed to determine DNA content showed a 30% increase in G1/G0 cells, which were transfected with axl RNAi compared to control. Altogether, these findings suggest an overexpression of Axl as part of a proliferative phenotype in RCC.