Mutational inactivation of the retinoblastoma gene (RB) is found in all retinoblastomas and in a subset of other human neoplasms, including sarcomas of bone or soft tissue and carcinomas of lung or breast. Exogenous copies of wild-type RB have been shown to suppress the tumorigenicity of several types of tumor cells with endogenous RB mutations, including a previously described human prostatic carcinoma cell line. To further support a role for RB inactivation in the genesis ofprostate cancer, seven primary or metastatic prostate carcinoma specimens were examined for evidence of RB mutation. By the use of immunoblot analysis and immunostaining of histologic sections, RB-encoded protein was readily detected in tumor cells offive specimens, was equivocally detected in one specimen, and was apparently absent from tumor cells of one specimen. RB mutations in the latter case were precisely characterized as (I) a deletion of 103 nucleotides containing transcriptional start sites and (it) loss of the second RB allele.The 103-base-pair deletion was sufficient to abolish the promoter activity of upstream DNA sequences in a heterologous expression system. These results (i) demonstrate thatRB can be inactivated in vivo by mutation of its promoter, (ii) confirm the existence ofRB mutations in some human prostate carcinomas, and (iii) suggest the use of immunohistochemical methods to screen for RB mutations in clinical samples of common adult neoplasms. mutations, as predicted by Knudson (9). RB mutations are also found in a subset of osteosarcomas, soft-tissue sarcomas, and carcinomas of breast and lung, suggesting a broad role for RB inactivation in the genesis of human tumors (8).Finally, restoration of the normal RB gene product, ppllORB, into pp11ORB-deficient (RB-) tumor cells alters several aspects of their neoplastic phenotype, including suppression of tumorigenicity in nude mice (10, 11). Because of these properties, RB is a model for studying other candidate tumor suppressor genes.In a previous study of three human prostate carcinoma cell lines, one was found to express a mutated RB mRNA and an abnormally small RB protein that was functionally inactive in tumor suppression (11). To establish the existence of RB mutations in native prostate carcinomas, primary or metastatic tumor specimens were screened for loss of RB protein expression. Two tumors were found to have severely reduced or undetectable amounts of RB protein by immunoblot analysis and by immunostaining of histologic sections. Direct detection of mutated RB alleles is hindered by the large size (';200 kilobases) and structural complexity (27 exons) of this gene (12). Nevertheless, a detailed genetic analysis of one tumor revealed both mutations leading to RB inactivation: (i) a 103-base-pair (bp) deletion that abolished activity of the RB promoter and (ii) loss of the second normal RB allele. Prostate carcinoma is the most common cancer in men (1).

NMR spectroscopy-based metabolomics still needs development in quantification procedures. A method was designed for quantitative two-dimensional high resolution magic angle spinning (HRMAS) proton-NMR spectroscopy-based metabolite profiling of intact cells. It uses referencing of metabolite-related NMR signals to protein-related NMR signals and yields straightforward and automatable metabolite profiling. The method enables exploitation of only two-dimensionally visible metabolites and combination of one- and two-dimensional spectra, thus providing an appreciable number of screened metabolites. With this procedure, 32 intracellular metabolites were attributed and quantified in human normal fibroblasts and tumor cells. The phenotype of several tumor cell lines (MCF7, PC3, 143B, and HepG2) was characterized by high levels of glutathione in cell lines with the higher proliferation rate, high levels of creatine, low levels of free amino acids, increased levels of phospholipid derivatives (mostly phosphocholine), and lower lactate content in cell lines with the higher proliferation rate. Other metabolites such as fatty acids differed widely among tumor cell lines. The response of tumor cell lines to chemotherapy also was evaluated by differential metabolite profiling, bringing insights into drug cytotoxicity and tumor cell adaptive mechanisms. The method may prove widely applicable to tumor cell phenotyping.

The design, synthesis, and screening of dual PI3K/mTOR inhibitors that gave nanomolar enzymatic and cellular activities on both targets with an acceptable kinase selectivity profile are described. A docking study was performed to understand the binding mode of the compounds and to explain the differences in biological activity. In addition, cellular effects of the best dual inhibitors were determined on six cancer cell lines and compared to those on a healthy diploid cell line for cellular cytotoxicity. Two compounds are highly potent on cancer cells in the submicromolar range without any toxicity on healthy cells. A more detailed analysis of the cellular effect of these PI3K/mTOR dual inhibitors demonstrated that they induce G1-phase cell cycle arrest in breast cancer cells and trigger apoptosis. These compounds show an interesting kinase profile as dual PI3K/mTOR tool compounds or as a chemical series for further optimization to progress into in vivo experiments.

In sporadic breast cancer, no mutations of the BRCA1 gene have been reported so far, whereas BRCA1 mRNA is markedly decreased in invasive breast cancer. To elucidate the contribution of the BRCA1 gene in sporadic breast cancer, we quantified the BRCA1 protein, using [ 125 I] labeling of wholecell proteins, lentil-lectin affinity chromatography, immunoprecitation by anti-BRCA1 antibodies (C-20, D-20, I-20 and K-18), purification of the immune complex by protein A affinity chromatography and chromato-focusing. As loss of 1 allele may lead to a decreased expression of the gene, 10 tumors were previously checked for loss of heterozygosity (LOH) of the BRCA1 gene, using 3 intragenic microsatellite markers. Our results indicated that the BRCA1 gene product was decreased in the 4 tumors with LOH compared with matched normal breast tissues. Reduced amounts of BRCA1 protein were also detected in 3 of 6 tumors without LOH. Our quantitative method allowed us to demonstrate that the BRCA1 protein level was decreased in sporadic invasive breast carcinomas with or without LOH of the BRCA1 gene, implying multiple mechanisms of BRCA1 expression downregulation in these tumors. Our data suggest that the amount of BRCA1 protein present may play an important role in human sporadic breast carcinoma. Int. J. Cancer 80:823-826, 1999. Wiley-Liss, Inc.While familial breast cancer or familial breast/ovarian cancer often accompanies germ-line mutations of the BRCA1 gene (Miki et al., 1994), there is no evidence of BRCA1 mutations in sporadic breast cancer and BRCA1 mutations in sporadic ovarian cancer are rare (Futreal et al., 1994, Merajver et al., 1995. However BRCA1 gene transcription is markedly reduced in invasive breast tumors relative to normal breast epithelium, ductal carcinoma in situ (Thompson et al., 1995) and breast cancer cell lines (Ribieras et al., 1997), suggesting a role for BRCA1 in sporadic tumors. So far, BRCA1 gene expression in tumor tissues and cell lines has been monitored mainly at the mRNA level, by either Northern blot (Marquis et al., 1995), ribonuclease protection assay (Thompson et al., 1995) or competitive RT-PCR (Ribieras et al., 1997). In this study, we evaluated the relative amount of BRCA1 protein in ductal invasive carcinomas, using a strategy of biochemical purification previously devised in our laboratory for measureing HLA-DR antigen amounts (Bernard et al., 1984(Bernard et al., , 1985 and adapted for the BRCA1 protein (Bernard-Gallon et al., 1997).As loss of 1 allele might lead to reduced expression of the corresponding gene, we searched for loss of heterozygosity (LOH) of the BRCA1 gene, using 3 intragenic microsatellite markers. The BRCA1 protein was then quantified in the 2 groups of tumors, with and without LOH, and compared in tumors and normal matched tissues. Our results show that the amount of BRCA1 protein was decreased in all tumors with LOH as well as in a majority of ductal invasive carcinomas without LOH. MATERIAL AND METHODS Patient samplesTen sporadic invasive ductal breast carcinomas...