Developmental expression and assembly of connexins into homomeric and heteromeric gap junction hemichannels in the mouse mammary gland.

During the development of the mammary gland, duct-lining epithelial cells progress through a program of expansive proliferation, followed by a terminal differentiation that allows for the biosynthesis and secretion of milk during lactation. The role of gap junction proteins, connexins, in the development and function of this secretory epithelium was investigated. Connexins, Cx26 and Cx32, were differentially expressed throughout pregnancy and lactation in alveolar cells. Cx26 poly-(A)(+) RNA and protein levels increased from early pregnancy, whereas Cx32 was detectable only during lactation. At this time, immunolocalization of connexins by confocal microscopy and immunogold labeling of high-pressure frozen freeze-substituted tissue showed that both connexins colocalized to the same junctional plaque. Analysis of gap junction hemichannels (connexons) isolated from lactating mammary gland plasma membranes by a rate-density centrifugation procedure, followed by immunoprecipitation and by size-exclusion chromatography, showed that Cx26 and Cx32 were organized as homomeric and heteromeric connexons. Structural diversity in the assembly of gap junction hemichannels demonstrated between pregnant and lactating mammary gland may account for differences in ionic and molecular signaling that may physiologically influence the onset and/or maintenance of the secretory phenotype of alveolar epithelial cells.

Interaction of axin and Dvl-2 proteins regulates Dvl-2-stimulated TCF-dependent transcription.

Axin promotes the phosphorylation of beta-catenin by GSK-3beta, leading to beta-catenin degradation. Wnt signals interfere with beta-catenin turnover, resulting in enhanced transcription of target genes through the increased formation of beta-catenin complexes containing TCF transcription factors. Little is known about how GSK-3beta-mediated beta-catenin turnover is regulated in response to Wnt signals. We have explored the relationship between Axin and Dvl-2, a member of the Dishevelled family of proteins that function upstream of GSK-3beta. Expression of Dvl-2 activated TCF-dependent transcription. This was blocked by co-expression of GSK-3beta or Axin. Expression of a 59 amino acid GSK-3beta-binding region from Axin strongly activated transcription in the absence of an upstream signal. Introduction of a point mutation into full-length Axin that prevented GSK-3beta binding also generated a transcriptional activator. When co-expressed, Axin and Dvl-2 co-localized within expressing cells. When Dvl-2 localization was altered using a C-terminal CAAX motif, Axin was also redistributed, suggesting a close association between the two proteins, a conclusion supported by co-immunoprecipitation data. Deletion analysis suggested that Dvl-association determinants within Axin were contained between residues 603 and 810. The association of Axin with Dvl-2 may be important in the transmission of Wnt signals from Dvl-2 to GSK-3beta.

Compartment switching of WNT-2 expression in human breast tumors.

WNT-2 is a secreted polypeptide with mitogenic effects in murine mammary epithelial cells, but its role in human cancer is unknown. Using RNase protection analysis of primary cell preparations and in situ hybridization analysis, we report that WNT-2 is expressed at low levels in normal human breast fibroblasts but not in epithelial cells. WNT-2 was found to be expressed at high levels in both the epithelium and stroma of 5 of 11 infiltrating carcinomas and 2 of 6 fibroadenomas. The high level of WNT-2 expression in tumor epithelium suggests that tumorigenesis may involve the ectopic expression of WNT-2 and the creation of an autocrine Wnt signaling loop.

Abnormalities of the p53 MDM2 and DCC genes in human leiomyosarcomas.

In this study we have screened a series of 29 primary leiomyosarcomas for abnormalities of both the p53 gene and the MDM2 gene, which encodes a p53-associated protein. SSCP (single-strand conformation polymorphism) analysis and direct sequencing of polymerase chain reaction (PCR)-amplified DNA were used to establish that 6/29 tumours possessed point mutations of the p53 gene. Using a monoclonal antibody that recognises the p53 protein in immunohistochemical staining experiments, we observed overexpression of the p53 protein in five of the six tumours containing point mutations in the p53 gene. Southern analysis of tumour DNA revealed that 2/29 tumours demonstrated amplification of the MDM2 gene. When considered together, these results indicate that alterations in both the p53 gene and MDM2 gene are important in the development of a significant minority of leiomyosarcomas. In addition, we have demonstrated a significant association between the presence of abnormalities of the p53 gene or MDM2 genes in leiomyosarcomas and a more advanced clinicopathological stage (P = 0.03). We have also examined the role of the DCC tumour-suppressor gene in the development of human soft-tissue tumours in a variety of histological types. Except for evidence of a rearrangement in a single leiomyosarcoma cell line, SK-UT-1, we have found no direct evidence to support a role for mutation of the gene in the development of human soft-tissue tumours.

Expression of BCL-2 in primary breast cancer and its correlation with tumour phenotype. For the International (Ludwig) Breast Cancer Study Group.

BACKGROUND: The role of apoptosis (programmed cell death) in the development and progression of breast cancer is unknown. Recently the bcl-2 gene has been shown to block apoptosis and thus may promote tumour development. BCL-2 is localized to the luminal cells of the normal breast, which are considered to be the origin of malignant breast disease. PATIENTS AND METHODS: Immunocytochemistry using anti bcl-2- antibody was performed on 107 breast cancer specimens belonging to node-positive patients from the Ludwig Breast Cancer Studies I-IV and the results were correlated with survival, tumour grade, S-phase, oestrogen and progesterone receptor status and c-erb B-2 expression. Western and Southern blotting together with immunofluorescence were performed on the breast cancer cell lines BT-20, BT-474, MDA-MB-361, T47-D and MCF-7. RESULTS: In the breast cancer derived cell line MCF-7 BCL-2 is expressed to a level similar to that of the B-lymphoma cell line Karpas 231 with t(14;18)(q32.3;q21.3), but no evidence of a rearrangement or gene amplification was identified. In a study of 107 breast cancers from the International Breast Cancer Study Group Trials I-IV we have demonstrated a very significant inverse correlation of BCL-2 with c-erbB-2 expression (p = 0.002), and a positive correlation with oestrogen receptors (p = 0.001) and progesterone receptors (p = 0.05). In this study there was no correlation of expression with S-phase fraction in the tumours or with any stage in the cell cycle as assessed in MCF-7 cells. CONCLUSION: We conclude that BCL-2 might contribute to the malignant phenotype of breast cancer by modulation of biological behaviour of cancer cells.

Expression of p53 in premalignant and malignant squamous epithelium.

Using three antibodies (JG8, CM-1 and 1081) directed to the p53 protein, strong positivity was found in 16/47 (34.0%) of mucosal squamous cell carcinomas of the head and neck and in two squamous carcinoma cell lines (LICR-LON- HN5 and HN6Rr). The presence of the mutant p53 was confirmed in the cell lines as substitutions in exon 7 (codon 238, TGT greater than AGT) and exon 5 (codon 152, CCG greater than CTG) respectively. Positive staining was seen only in the undifferentiated cells and progressively lost as the cells keratinized, both in the tumour specimens and in the cell lines. Similar results were seen in areas of dysplasia, well removed from the site of the primary tumour. Staining of epidermal lesions showed positivity in 2/12 (16.6%) cases of Bowen's disease, 0/12 (0.0%) cases of solar keratosis, 0/10 (0.0%) basal cell carcinomas and in 3/20 (15.0%) squamous cell carcinomas. These results are discussed in relation to the multifocal origin of squamous cell carcinomas, the role of p53 mutations in squamous cell carcinomas from different sites and the significance of the 'basal' distribution of p53 as a normal growth regulator. The possible significance of the distribution of p53 in squamous epithelium as it relates to papilloma virus infection is also considered.

Growth factor stimulation of proliferating cell nuclear antigen (PCNA) in human breast epithelium in organ culture.

Normal human breast explants were maintained in serum-free culture for 7 days in the presence of either insulin hydrocortisone and cholera toxin (I/H/CT), epidermal growth factor (EGF) or transforming growth factor-alpha (TGF-alpha). Explants were labelled with [3H] thymidine, fixed in methacarn and processed for autoradiography. Parallel sections were immunolabelled with anti-PCNA antibody and analysed with a CAS 200 image analyser. Thymidine labelling index (TLI) and PCNA expression produced similar results with both indices increased in response to I/H/CT, EGF and TGF-alpha. In sections double labelled for PCNA and autoradiography the majority of labelled cells were positive for both markers.