Expression of circadian clock genes and proteins in urothelial cancer is related to cancer-associated genes.

BACKGROUND: The purpose of this study was to evaluate invasive and metastatic potential of urothelial cancer by investigating differential expression of various clock genes/proteins participating in the 24 h circadian rhythms and to compare these gene expressions with transcription of other cancer-associated genes. METHODS: Twenty seven paired samples of tumour and benign tissue collected from patients who underwent cystectomy were analysed and compared to 15 samples of normal bladder tissue taken from patients who underwent cystoscopy for benign prostate hyperplasia (unrelated donors). Immunohistochemical analyses were made for clock and clock-related proteins. In addition, the gene-expression levels of 22 genes (clock genes, casein kinases, oncogenes, tumour suppressor genes and cytokeratins) were analysed by real-time quantitative PCR (qPCR). RESULTS: Considerable up- or down-regulation and altered cellular distribution of different clock proteins, a reduction of casein kinase1A1 (CSNK1A1) and increase of casein kinase alpha 1 E (CSNK1E) were found. The pattern was significantly correlated with simultaneous up-regulation of stimulatory tumour markers, and a down-regulation of several suppressor genes. The pattern was mainly seen in aneuploid high-grade cancers. Considerable alterations were also found in the neighbouring bladder mucosa. CONCLUSIONS: The close correlation between altered expression of various clock genes and common tumour markers in urothelial cancer indicates that disturbed function in the cellular clock work may be an important additional mechanism contributing to cancer progression and malignant behaviour.

Time-dependent biological differences in molecular markers of high-grade urothelial cancer over 7 decades (ras proteins, pTEN, uPAR, PAI-1 and MMP-9).

The aim of this study was to evaluate changes and correlations between various molecular markers related to growth regulation and invasiveness in urothelial carcinomas in samples collected from 1932 to 2004. Paraffin-embedded autopsy/biopsy tissues from 144 patients were stained with antibodies against H-K-N ras proteins, pTEN protein, urokinase plasminogen activator receptor (uPAR), plasminogen activator inhibitor-1 (PAI-1) and matrix metalloproteinase-9 (MMP-9) and analyzed by in situ hybridization. Statistical analysis was performed by SPSS using cross tabulation and logistic regression. While the presence of K-ras, N-ras, PAI-1, and loss of pTEN increased over the last few decades, uPAR expression decreased during the same period. The increase in K-ras expression associated positively with the increase in expression of the other two ras proteins, H-ras and N-ras, and the loss of pTEN. A strong positive correlation was also observed between PAI-1 and uPAR, PAI-1 and previously detected markers, EGFR (epidermal growth factor receptor) and p53. Presence of uPAR was found to be positively associated with p16 expression. Multivariate analysis with clinical parameters revealed a positive correlation between PAI-1 expression and tumour grade, CkHMW (high molecular weight cytokeratin) and tumour grade, CkHMW and metastasis, EGFR and metastasis. mRNA could be detected in samples from the last 50 years while older samples were negative, indicating its complete degradation during longer storage. In conclusion, increased accumulation of K-ras, N-ras, and PAI-1 together with loss of pTEN in bladder carcinomas of grades II and III seems to be more dominant in recent times, suggesting an altered malignant potential in these neoplasms.

Immunohistochemical markers in urinary bladder carcinomas from paraffin-embedded archival tissue after storage for 5-70 years.

OBJECTIVE: To evaluate archival tissue specimens from bladder tumours and seek molecular changes in samples collected over seven decades previously, as although the frequencies of some cancer types have remained stable during the last 50 years, the incidence of others, including bladder tumours, has increased significantly, and molecular analyses of bladder cancer over periods with an increasing incidence are of interest as the findings might reflect varying external influences. MATERIALS AND METHODS: Immunohistochemical staining with the biological markers p53 protein, p16 protein, epidermal growth factor receptor (EGFR), cytokeratin 7 and high molecular weight 34betaE12 cytokeratin (HMW-cytokeratin, characteristic of basal cells) was used on archival, paraffin wax-embedded autopsy/biopsy tissue material collected from 144 patients with invasive bladder cancer (World Health Organisation grade II and III). The cases were selected from the periods 1932-48, 1950-59, 1960-70 and 1990-2004. Control immunohistochemistry was done on available normal tissue (i.e. connective and fatty tissue, heart, lungs and normal urinary bladder epithelium) obtained from the autopsies. RESULTS: The normal tissues were all largely negative for EGFR, had <1% positively stained nuclei for p53 and strong positive reactions for p16, and in epithelial tissues the two cytokeratins were detected. The positive scores for HMW-cytokeratin in the tumour tissue decreased significantly from approximately 90% to 30% over the 70 years. For p53 there was a higher fraction of positive scores (borderline significant) with time. The p16-positive tumours showed no significant variation, with the highest frequency of positive scores in recent years. Overexpression of EGFR in the tumours was significantly correlated with the occurrence of HMW-cytokeratin and decreased from approximately 85% to 65% (not significant), with the lowest frequency in the samples from 1990 to 2004. Autolysis after death or long storage periods did not compromise good quality in the histochemical analyses of the autopsy tissue. CONCLUSION: The higher frequency of HMW-cytokeratin, lower p53 accumulation and more EGFR expression in grade II and III urinary bladder carcinomas from the 1930s could indicate different phenotypes in bladder cancer during this 70-year period. The successful detection of these protein markers in old archival material allows larger retrospective studies that might increase the understanding of molecular carcinogenesis in bladder cancer.