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The reduction or loss of plakoglobin expression in late-stage bladder cancer has been correlated with poor survival where upregulation of this catenin member by histone deacetylase inhibitors has been shown to accompany tumour suppression in an in vivo model. In this study, we directly addressed the question of the role of plakoglobin in bladder tumorigenesis following restoration, or knockdown of expression in bladder carcinoma cell lines. Restoration of plakoglobin expression resulted in a reduction in migration and suppression of tumorigenic potential in vivo. Immunocytochemistry revealed cytoplasmic and membranous localisation of plakoglobin in transfectants with

Aims—The relation between lobular carcinoma in situ (LCIS) and invasive breast cancer is unresolved. In an attempt to establish whether LCIS is a precursor of invasive cancer the mutational status and the expression of E-cadherin was analysed in LCIS and associated invasive breast carcinoma in 23 patients. Methods—Foci of LCIS and associated invasive carcinoma were individually microdissected from tissue from 23 patients. Exons 4–16 of the E-cadherin gene were analysed using single strand conformation polymorphism (SSCP); protein expression and the localisation of E-cadherin and β-catenin were assessed with the use of immunohistochemistry. Results—Immunohistochemistry revealed a lack of expression of E-cadherin and β-catenin in most LCIS samples and invasive foci. In all but four cases, the staining pattern was identical in the LCIS and associated invasive areas. When E-cadherin was absent, β-catenin was also undetected, suggesting a lack of expression of alternative classic cadherin members in these lesions. Coincident E-cadherin mutations in LCIS and associated invasive carcinoma were not identified in this series of patients. However, mutational analysis of E-cadherin in multiple foci of carcinoma in situ surrounding an invasive lesion provided evidence to support ductal carcinoma in situ as a precursor of invasive ductal carcinoma. Conclusion—These data support the hypothesis that LCIS is not a precursor of invasive breast carcinoma but a marker of increased risk of developing invasive disease.

Introduction of a H-ras oncogene into an SV-40 immortalized human urothelial cell lines (SV-HUC) results in morphologically altered cell clones which acquire tumorigenic potential following serial passaging in culture. Early and late passage cells, from individual ras transfected clones exhibiting different tumorigenic potential, display increased growth factor synthesis in mitogenic assays. Northern blot analysis revealed induction of TGF-alpha mRNA concomitant with the introduction of a H-ras oncogene with no modulation in EGF receptor expression observed throughout neoplastic progression. Consistent with completion of an autocrine loop, down modulation and activation of EGF receptors was observed in early passage cells coincident with TGF-alpha expression. In this human urothelial progression model TGF-alpha secretion follows the introduction of a H-ras oncogene prior to the acquisition of tumorigenic potential.

In this study, we report the use of a panel of four ras antibodies in Western blot analysis which characterize the expression of individual ras members in different cell populations. The specificity of the different antibodies to H-, N- or K-ras proteins was established in a panel of NIH/3T3 transfectants and human cell lines harboring known ras oncogenic events. Localization of individual ras members in one dimensional gel electrophoresis revealed slower migration of K-ras p21 products, whilst H- and N-ras proteins co-migrated in this system. Identification of migrational differences between activated proto-oncogene products was facilitated using ras member specific probes. Application of this approach to normal gastrointestinal mucosa.revealed over-representation of K-ras p21 in both stomach and colon epithelium in the rat. This differential ras profile was maintained in normal human colon mucosa and colon tumors from the same individual. Use of such antibodies now enables dissection of ras proto-oncoprotein expression in different tissues and compartments within tissue with the ability to characterize ras mutational events in neoplastic progression.

In this study we report detection of mdr1 gene expression in the liver metastases of 7/11 patients with colon carcinoma and characterise the MDR phenotype associated with a panel of 19 human colon carcinoma cell lines. Within this panel, mdr1 mRNA biosynthesis and surface localisation of Pgp were assessed with respect to MDR functionality where the cell lines are representative of different clinical stages of tumour progression, metastatic potential and differentiation. The data indicates that constitutive levels of mdr1 mRNA/Pgp expression may not necessarily result in the functional expression of the MDR phenotype. While low levels of mdr1 mRNA/Pgp were detected in 5/8 well differentiated colon cell lines, only 2/8 were functionally MDR. In contrast, 10/11 moderate and poorly differentiated lines expressed mdr1 mRNA/Pgp and of these, 9/11 were functionally MDR. The phosphorylation status of the mature 170 kD P-glycoprotein and the surface localisation of this glycoprotein showed the strongest correlation with functionality. Analysis of cell lines for cross-resistance and chemosensitivity profiles against a battery of chemotherapeutic drugs suggests multiple mechanisms, in addition to Pgp, contribute to the overall resistance of colorectal cancer. Images Figure 1 Figure 2 Figure 3 Figure 4

Analysis of human tumour-derived cell lines has previously resulted in the identification of novel transformation-related elements and provided a useful tool for functional studies of different genes. To establish the utility of such cell lines as indicators of change relevant to urothelial cancer, we have characterised the expression of five genes (p53, MDM2, Rb, E-cadherin, APC) within a panel of human bladder carcinoma cell lines. Using single-strand conformation polymorphism (SSCP) and direct sequencing, p53 mutations were identified in 7/15 (47%) cell lines reflecting events reported in bladder tumours. Immunohistochemical analysis of p53 in cultured cells and in paraffin-embedded sections of xenografts from the cell line panel revealed discordant results. An absence of p53 nuclear staining was associated with an exon 5 mutation in EJ and with multiple p53 mutations found in J82. Two cell lines positive for p53 staining in the absence of detectable mutation displayed overexpression of MDM2 (PSI, HT1197) in Western blot analysis. Loss or aberrant Rb expression was recorded in 5/15 (TCCSUP, SCaBER, 5637, HT1376, J82) cell lines. Absence of E-cadherin was recorded in 5/15 cell lines (TCCSUP, EJ, KK47, UM-UC-3, J82) with loss of alpha-catenin in immunoprecipitated E-cadherin complexes of CUBIII. Western blot analysis of APC revealed a truncated protein in 1/15 (CUBIII) cell lines. The characterisation of oncogenic events within this panel of human bladder carcinoma cell lines establishes a representation of change observed in bladder tumours and better defines the genotypic background in these experimental human cell models of neoplastic progression. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6

In an established colon differentiation model, introduction of a c-H-ras-1 oncogene into a poorly differentiated human colon carcinoma cell line (Clone A) results in changes associated with the acquisition of a more differentiated phenotype. Down-regulation of mdr-1 mRNA was shown to accompany ras-related differentiation events resulting in decreased Pgp synthesis and a significant reduction in membrane Pgp as detected by immunoprecipitation, Western-blot and FACS analysis. Consistent with these observations was a reduction in Pgp-mediated drug resistance associated with Clone-A ras transfectants, with no alteration in drug sensitivity being observed with non-MDR drugs in these cells. An alternative differentiation model involves exposure of Clone-A cells to sodium butyrate. Under these conditions, differentiation-related changes resulted in up-regulation of mdr-1 mRNA and Pgp synthesis, although no alteration in drug sensitivity was recorded. In agreement with this observation, the levels of membrane-associated Pgp remained unchanged throughout the period of exposure to sodium butyrate. This study shows that modulation of Pgp expression in colon differentiation is dependent upon the differentiation induction agent used.