Germline mutations of the tumour suppressor gene BRCA1 are involved in the predisposition and development of breast cancer and account for 20 -45% of all hereditary cases. There is an increasing evidence that these tumours are characterised by a specific phenotype and pattern of gene expression. We have hypothesised that differences in chemosensitivity might parallel molecular heterogeneity of hereditary and sporadic breast tumours. To this end, we have investigated the chemosensitivity of the BRCA1-defective HCC1937 breast cancer cell line, and the BRCA1-competent MCF-7 (hormone-sensitive) and MDA-MB231 (hormoneinsensitive) breast cancer cell lines using the MTT assay. The 50% inhibitory concentration (IC 50 ) for the individual compounds were derived by interpolate plot analysis of the logarithmic scalar concentration curve after a 48 h exposure. HCC1937 cells were significantly (Po0.005) more sensitive to cisplatin (CDDP) (IC 50 : 30-40 mM) compared with MCF-7 (IC 50 : 60-70 mM) and MDA-MB231 (IC 50 : 90 -100 mM) cells. On the other hand, BRCA1-defective breast cancer cells were significantly less sensitive to doxorubicin (Dox) (IC 50 : 45-50 mM) compared with MCF-7 (IC 50 : 1-5 mM) and MDA-MB231 (IC 50 : 5-10 mM) (Po0.02), as well as to paclitaxel (Tax) (IC 50 : 42 mM for HCC1937, 0.1 -0.2 mM for MCF-7 and 0.01 -0.02 mM for MDA-MB231) (Po0.001). Full-length BRCA1 cDNA transfection of BRCA1-defective HCC1937 cells led to the reconstituted expression of BRCA1 protein in HCC1937/ WT BRCA1-derived cell clone, but did not reduce tumour cell growth in soft agar. BRCA1 reconstitution reverted the hypersensitivity to CDDP (Po0.02), and restored the sensitivity to Dox (Po0.05) and Tax (Po0.001), compared with parental HCC1937 cells. Taken together, our findings suggest a specific chemosensitivity profile of BRCA1-defective cells in vitro, which is dependent on BRCA1 protein expression, and suggest prospective preclinical and clinical investigation for the development of tailored therapeutical approaches in this setting.
Hereditary nonpolyposis colon cancer (HNPCC) is associated with malfunction of postreplicative mismatch repair (MMR). While a majority of HNPCC-associated mutations in the MMR genes MLH1, MSH2, or MSH6 genes cause truncations-and thus loss of function--of the respective polypeptides, little is currently known about the biochemical defects associated with nontruncating mutations. We studied the interactions of six MLH1 variants, carrying either missense mutations or in-frame deletions, with normal PMS2 and tested the functionality of these heterodimers of MLH1 and PMS2 (MutL(alpha)) in an in vitro MMR assay. Three MLH1 carboxy-terminal mutations, consisting of internal deletions of exon 16 (amino acids 578-632) or exon 17 (amino acids 633-663), or a missense R659P mutation in exon 17, affected the formation of a functional MutL(alpha). Interestingly, mutations C77R and I107R in the amino-terminal part of MLH1 did not affect its heterodimerization with PMS2. The complexes MLH1(C77R)/PMS2 and MLH1(I107R)/PMS2, however, failed to complement a MMR-deficient extract lacking a functional MutL(alpha). As all these five mutations were identified in typical HNPCC families and produce nonfunctional proteins, they can be considered disease-causing. In contrast, the third amino-terminal mutation S93G did not affect the heterodimerization, and the MLH1(S93G)/PMS2 variant was functional in the in vitro MMR assay, given thus the nature of the HNPCC family in question. Although the missense mutation segregates with the disease, the mean age of onset in the family is unusually high (approximately 65 years).
Transcription of the H ferritin gene in vivo is stimulated by cAMP and repressed by the E1A oncoprotein. We report here the identification of the cis-element in the human promoter responsive to both cAMP-and E1A-mediated signals. This promoter region is included between positions ؊62 to ؊45 and binds a approximate 120-kDa transcription factor called Bbf. Bbf forms a complex in vivo with the coactivator molecules p300 and CBP. Recombinant E1A protein reduces the formation of these complexes. In vivo overexpression of p300 in HeLa cells reverses the E1A-mediated inhibition of the ferritin promoter transcription driven by Bbf. These data suggest the existence of a common mechanism for the cAMP activation and the E1A-mediated repression of H ferritin transcription.
Several genes have been involved in the pathogenesis of hereditary breast/ovarian cancer (BOC), but mutations in the BRCA1 gene are by far the most recurrent. In this study, we report the identification of a founder mutation in a geographically and historically homogeneous population from Calabria, a south Italian region. A screening performed on 24 patients from unrelated families highlighted the high prevalence of a 5083del19 alteration in the BRCA1 gene, which accounts for 33% of the overall gene mutations. The same mutation was also detected in 4 patients, all of Calabrian origin, referred to us by research centres from the north of Italy. Allelotype analysis, performed on probands and unaffected family members revealed the presence a common allele, therefore suggesting a founder effect due to a common ancestor. Our findings underscore the importance of ethnic background homogeneity in patients' selection and highlight the usefulness of founder mutations as a potential tool for optimisation of preclinical diagnosis in gene carriers and therapeutic approaches in affected individuals.
The ability of pathogens to sequester iron from their host cells and proteins affects their virulence. Moreover, iron is required for various innate host defense mechanisms as well as for acquired immune responses. Therefore, intracellular iron concentration may influence the interplay between pathogens and immune system. Here, we investigated whether changes in iron concentrations and intracellular ferritin heavy chain (FTH) abundance may modulate the expression of Major Histocompatibility Complex molecules (MHC), and susceptibility to Natural Killer (NK) cell cytotoxicity. FTH downregulation, either by shRNA transfection or iron chelation, led to MHC surface reduction in primary cancer cells and macrophages. On the contrary, mouse embryonic fibroblasts (MEFs) from NCOA4 null mice accumulated FTH for ferritinophagy impairment and displayed MHC class I cell surface overexpression. Low iron concentration, but not FTH, interfered with IFN-γ receptor signaling, preventing the increase of MHC-class I molecules on the membrane by obstructing STAT1 phosphorylation and nuclear translocation. Finally, iron depletion and FTH downregulation increased the target susceptibility of both primary cancer cells and macrophages to NK cell recognition. In conclusion, the reduction of iron and FTH may influence the expression of MHC class I molecules leading to NK cells activation.
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