An interaction between apolipoprotein E and TERE1 with a possible association with bladder tumor formation.

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.

Alterations in tropomyosin isoform expression in human transitional cell carcinoma of the urinary bladder.

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.

Expression of the proto-oncogene Axl in renal cell carcinoma.

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.

TERE1, a novel gene affecting growth regulation in prostate carcinoma.

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.

KIAA1096, a gene on chromosome 1q, is amplified and overexpressed in bladder cancer.

Chromosomal gain on 1q23-24 is a cytogenetic finding found in approximately 30% of bladder tumors. Currently, no defined or candidate tumor-associated genes from this region have been identified. The objective of this study was to identify and quantitate the expression of putative cancer genes located at this chromosome locus in normal urothelium, superficial, and muscle invasive bladder tumors. We examined both normal and bladder cancer tissue specimens (N = 40-80 RNA, DNA, and protein) by semiquantitative RT/PCR, genomic PCR, and by Western blotting. The KIAA1096 gene is located at 1q23-24 with no overexpression or amplification in normal urothelium, but was significantly upregulated in 30% of tumors (P = 0.0001). There was a trend towards increased expression in invasive compared to superficial lesions (P = 0.06). A significant increase in gene copy was also found in a 38% of TCC of the bladder compared to normal bladder mucosa or peripheral blood lymphocytes. Immunohistochemistry (IHC) demonstrated KIAA1096 expression in nonmalignant bladder mucosa tissue but apparent upregulation in invasive transitional cell carcinoma. Two other genes, CH1 and RGS5, which are situated in the same region of chromosome 1q, demonstrated disparate patterns of expression. In summary, KIAA1096 is a gene situated at 1q23-24, which demonstrated a pattern of RNA and DNA expression consistent with the 38% expression of cytogenetic amplification noted on previous studies. This gene may, therefore, be a putative marker for this cytogenetic phenomenon and provide an opportunity to evaluate the clinical significance of previous cytogenetic findings.

The isolation of an immortalized and tumorigenic cell line from p21WAF1 null mouse bladders.

Given a role for the deregulation of p21WAF1 in the progression of bladder tumors, we examined the growth of cultured urothelial cells from wild-type and p21WAF1 null bladders. Bladders were excised, minced from euthanized p21WAF1 and wild-type mice, treated overnight with dispase, and then placed into flasks coated with collagen type I in Dulbecco modified Eagle medium with 10% fetal calf serum. After an overnight incubation, the media was replaced with a serum-free media and a portion of explants were treated with 12-O-tetrade-canoylphorbol-13-acetate (TPA) on day 7 and continued for either 4 or 9 wk. The urothelial origin of any surviving epithelial cells was determined by reverse transcription-polymerase chain reaction (RT-PCR) using uroplakin II-specific primers, and the expression of the cell cycle-related proteins, p16INK4 and p19ARF, was examined by semiquantitative RT-PCR and Western blotting. Isolated wild-type and serially passaged p21WAF1 null epithelial-like cells were then injected subcutaneously into nude mice. We found that phorbol ester treatment at two different concentrations significantly enhanced uroepithelial colony formation from isolated wild-type mouse bladder tissue. On the other hand, significantly fewer urothelial colonies were derived from p21WAF1 null bladder cells treated with phorbol ester. Although there was apparent senescence and cell death of epithelial foci and stromal cells in phorbol ester-treated and -untreated p21WAF1 null cultures, after 3 mo there was an apparent subpopulation of epitheloid cells that overgrew each flask. There was a significant decrease in the number of these serially passaged cells in the G1 phase of the cell cycle when compared with initial explant wild-type or p21WAF1 null cells. This subpopulation of epitheloid cells expressed the mouse uroplakin II gene, indicating a urothelial phenotype, but did not express either the p16INKa or p19ARF proteins, whereas p21WAF1 null bladders express both proteins. There was also a high level of expression of the p53 protein and a significant decrease in the expression of the p19ARF transcript in both p21WAF1 null bladder and p21WAF1 null cells. These p21WAF1 null cells could be easily passaged and when injected subcutaneously into nude mice, large tumors developed. Therefore, it appears that a subpopulation of urothelial cells from the p21WAF1 null bladder can develop a tumorigenic phenotype in vitro.