Novel markers for differentiation of lobular and ductal invasive breast carcinomas by laser microdissection and microarray analysis

BACKGROUND: Invasive ductal and lobular carcinomas (IDC and ILC) are the most common histological types of breast cancer. Clinical follow-up data and metastatic patterns suggest that the development and progression of these tumors are different. The aim of our study was to identify gene expression profiles of IDC and ILC in relation to normal breast epithelial cells. METHODS: We examined 30 samples (normal ductal and lobular cells from 10 patients, IDC cells from 5 patients, ILC cells from 5 patients) microdissected from cryosections of ten mastectomy specimens from postmenopausal patients. Fifty nanograms of total RNA were amplified and labeled by PCR and in vitro transcription. Samples were analysed upon Affymetrix U133 Plus 2.0 Arrays. The expression of seven differentially expressed genes (CDH1, EMP1, DDR1, DVL1, KRT5, KRT6, KRT17) was verified by immunohistochemistry on tissue microarrays. Expression of ASPN mRNA was validated by in situ hybridization on frozen sections, and CTHRC1, ASPN and COL3A1 were tested by PCR. RESULTS: Using GCOS pairwise comparison algorithm and rank products we have identified 84 named genes common to ILC versus normal cell types, 74 named genes common to IDC versus normal cell types, 78 named genes differentially expressed between normal ductal and lobular cells, and 28 named genes between IDC and ILC. Genes distinguishing between IDC and ILC are involved in epithelial-mesenchymal transition, TGF-beta and Wnt signaling. These changes were present in both tumor types but appeared to be more prominent in ILC. Immunohistochemistry for several novel markers (EMP1, DVL1, DDR1) distinguished large sets of IDC from ILC. CONCLUSION: IDC and ILC can be differentiated both at the gene and protein levels. In this study we report two candidate genes, asporin (ASPN) and collagen triple helix repeat containing 1 (CTHRC1) which might be significant in breast carcinogenesis. Besides E-cadherin, the proteins validated on tissue microarrays (EMP1, DVL1, DDR1) may represent novel immunohistochemical markers helpful in distinguishing between IDC and ILC. Further studies with larger sets of patients are needed to verify the gene expression profiles of various histological types of breast cancer in order to determine molecular subclassifications, prognosis and the optimum treatment strategies

Microarray analysis of bicalutamide action on telomerase activity, p53 pathway and viability of prostate carcinoma cell lines

Bicalutamide is a non-steroidal anti-androgen commonly used in the treatment of prostate carcinoma. We analysed the transcriptional response to bicalutamide treatment with the aim of explaining the inhibition of telomerase in the androgen-sensitive cell line LNCaP and the effects of bicalutamide on the androgen-insensitive cell line DU145. Cells treated with 80 muM bicalutamide in steroid-depleted medium for 1 day were analysed in duplicate by Affymetrix Human Genome Focus Arrays. Response to bicalutamide in LNCaP cells was represented by downregulation of androgen-regulated genes, activation of the p53 pathway and inhibition of telomerase, which was associated with downregulation of v-myc avian myelocytomatosis viral oncogene homologue (MYC) and telomerase reverse transcriptase subunit. In DU145 cells we observed the influence of cell density on bicalutamide effectivity such that highly confluent cells showed lesser sensitivity than low confluent ones. In conclusion, we provide an explanation for telomerase inhibition after androgen receptor blockade in LNCaP cells and we also report activation of the p53 pathway in LNCaP cells and in-vitro sensitivity to bicalutamide of low confluent androgen-insensitive DU145 cells. These findings might have implications for both experimental and clinical research into prostate cancer. In particular, activation of the p53 pathway after treatment with 80 microM bicalutamide could justify usage of bicalutamide dosages higher than 150 mg daily in androgen-sensitive carcinoma therapy

Characterisation of epitopes of pan-IgG/anti-G3m(u) and anti-Fc monoclonal antibodies.

The characterisation of monoclonal antibodies (MAbs) and their epitopes is important prior to their application as molecular probes. In this study, Western blotting using IgG1 Fc and pFc' fragments was employed to screen seven MAbs before pepscan analysis to determine their reactivity to potentially linear epitopes. MAbs PNF69C, PNF110A, X1A11 and MAbs WC2, G7C, JD312, 1A1 detected epitopes within the C(H)3 and C(H)2 domains, respectively. However, only four MAbs showed pepscan profiles that highlighted likely target residues. In particular, MAbs PNF69C and PNF110A that have previously been characterised with pan-IgG and anti-G3m(u) specificity, detected the peptide motif 338-KAKGQPR-344 which was located within the N-terminal region of the C(H)3 domain. Furthermore the majority of residues were present in all four IgG subclasses. Consequently the peptide identified was consistent with the pan-IgG nature of these antibodies. By using PCImdad, a molecular display programme, this sequence was visualised as surface accessible, located in the C(H)2/C(H)3 inter-domain region and proximal to the residue arginine(435). It is speculated that this residue may be important for phenotypic expression of G3m(u) and specificity of these reagents. Pepscan analysis of MAbs G7C and JD312 (both pan-IgG) highlighted the core peptide sequence 290-KPREE-294, which was present in the C(H)2 domain and was common to all four IgG subclasses. PCImdad also showed this region to be highly accessible and was consistent with previous bioinformatic and autoimmune analysis of IgG. Overall these MAbs may serve as useful anti-IgG or anti-G3m(u) reagents and probes of immunoglobulin structure