Marguerite Cuny

DNA amplifications at 20q13 and MDM2 define distinct subsets of evolved breast and ovarian tumours.

DNA amplification seems to be particularly frequent in human breast tumours and has been associated with cancer evolution and aggressiveness. Recent data indicate that new events should be added to the list, such as the amplifications at chromosome 20q13 or the MDM2 gene. The present work aimed at determining the incidence and clinicopathological signification of these amplifications in a large series of breast and ovarian tumours. We tested 1371 breast and 179 ovarian tumours by Southern blotting and observed amplification of 20q13 in 5.4% breast and 2.8% ovarian carcinomas, whereas MDM2 was found amplified in 5.3% and 3.8% of breast and ovarian tumours respectively. MDM2 RNA expression levels were analysed in a subset of 57 breast tumours and overexpression was observed in 4/57 (7%) of the tumours. Elevated expression levels coincided with amplification of the gene. In breast cancer, 20q13 and MDM2 amplifications seem to define subsets of aggressive tumours. Indeed, 20q13 was correlated to axillary nodal involvement and occurred preferentially in younger patients (< 50 years). Furthermore, 20q13 correlated, as did MDM2 amplification, to aneuploidy. In parallel, we had also tested our tumour DNAs for amplification of CCND1, ERBB-2 and MYC, which made it possible to test for correlations with 20q13 or MDM2 amplifications. Whereas 20q13 showed a very strong correlation to CCND1 amplification, that of MDM2 was prevalent in MYC-amplified tumours. Interestingly, 20q13 and MDM2 amplifications showed some degree of correlation to each other, which may possibly be owing to the fact that both events occurred preferentially in aneuploid tumours. In ovarian cancer, no statistically significant correlation was observed. However, 20q13 amplification occurred preferentially in stage 3 tumours and MDM2 was correlated to ERBB-2 amplification. This may suggest that in ovarian tumours also, 20q13 and MDM2 amplifications occur in late or aggressive cancers.

Amplification of the BRCA2 Pathway Gene EMSY in Sporadic Breast Cancer Is Related to Negative Outcome

DNA amplification at band q13 of chromosome 11 is common in breast cancer, and CCND1 and EMS1 remain the strongest candidate genes. However, amplification patterns are consistent with the existence of four cores of amplification, suggesting the involvement of additional genes. Here we present evidence strongly suggesting the involvement of the recently characterized EMSY gene in the formation of the telomeric amplicon. EMSY maps at 11q13.5, 100 kb centromeric to the GARP gene, which has been mapped within the core of the distal amplicon. The EMSY protein was shown to interact with BRCA2 and has a role in chromatin remodeling. This makes EMSY a strong candidate oncogene for the 11q13.5 amplicon. DNA amplification was studied in a total of 940 primary breast tumors and 39 breast cancer cell lines. Amplification profiles were consistent with the EMSY-GARP locus being amplified independently of CCND1 and/or EMS1. EMSY RNA expression levels were studied along with those of five other genes located at 11q13.5 by real-time quantitative PCR in the 39 cell lines and a subset of 65 tumors. EMSY overexpression correlated strongly with DNA amplification in both primary tumors and cell lines. In a subset of 296 patients, EMSY amplification was found by both uni- and multivariate analyses to correlate with shortened disease-free survival. These data indicate that EMSY is a strong candidate oncogene for the 11q13.5 amplicon.

17q21-q25 aberrations in breast cancer: combined allelotyping and CGH analysis reveals 5 regions of allelic imbalance among which two correspond to DNA amplification.

Chromosome 17q is frequently rearranged in breast cancer. Allelotyping studies have proposed the existence of at least four regions of allelic imbalance (AI). Here we present a study combining allelotyping using 19 CA repeat markers mapping in the 17q21 – 25 region and molecular cytogenetics (CGH and FISH). Allelotyping was undertaken on 178 pairs of cognate tumor and normal DNA in order to determine the number of regions of AI and define the shortest overlaps. AI ranged from 34 – 54% of the informative cases according to the marker and, overall, 66% of the tumors presented AI at one of the markers tested. Analysis of the patterns of imbalances revealed at least five common regions of imbalance respectively defined by markers: D17S855, which is intragenic of BRCA1 (SRO 1), D17S1607 (SRO 2), D17S1855 (SRO 3), between D17S789 and D17S785 (SRO 4) and D17S784 (SRO 5). In order to characterize the nature of the genetic events revealed by allelotyping we performed CGH analysis on a subset of 43 tumors presenting variable patterns of imbalance. CGH showed that AI at 17q could represent four different types of genetic events: loss of chromosome 17, gain of 17q, gain of 17q22 – q24, loss of 17q11 – q21 and/or 17q25 – qter. Some of these anomalies could occur concomitantly within the same tumor. Since 35% of the tumors analysed by CGH presented gains, these data indicated that AI at 17q were not solely indicative of losses of genetic material and could also represent DNA amplification. Gains were most commonly observed in the 17q23 – q24 regions. This suggested that AI in SRO 2 and SRO 3 corresponded to DNA amplification. To assess this, we isolated BAC clones by PCR screening for markers D17S1607 and D17S1855 and used these in FISH experiments on six breast tumor cell lines and 14 breast cancer specimens. FISH results showed that both D17S1607 and D17S1855 were frequently involved in DNA amplification (8 – 30 copies). Altogether, our data show that allelotyping can be efficiently used in amplicon mapping. Clinico-pathological correlations indicated that imbalance at 17q preferentially occurred in high grade, PR- and ERBB2 amplified tumors.

Variation of bcl-2 expression in breast ducts and lobules in relation to plasma progesterone levels: overexpression and absence of variation in fibroadenomas

Some women with benign breast disease eventually develop breast cancer. The mammary gland undergoes tissue remodelling according to hormonal influences, involving a balance between quiescence, proliferation, and mechanisms of cell death. Proliferation and/or apoptotic events could therefore be investigated to help understand the mechanisms of benign lesion formation and identify mastopathies with a poor prognosis. bcl‐2 expression was analysed by immunohistochemistry in 75 benign mastopathies. Protein levels were quantitated with an image analyser in various epithelial structures on frozen sections, including adenoses, fibroadenomas, ductal epithelial hyperplasias, cysts, and apparently normal surrounding lobules and ducts. bcl‐2 levels were equivalent in apparently normal lobules and ducts, as well as in cysts and ductal hyperplasias. bcl‐2 staining was significantly higher in fibroadenomas, known to be of lobular origin [mean=10·1, quantitative immunochemistry score (QIC) arbitrary units (AU), n=19], than in normal lobules (mean=5·1 AU, n=43, P=7×10−5). bcl‐2 levels in normal lobules and ducts varied according to the menstrual cycle, being higher during the follicular than the luteal phase (P=1·8×10−2 and P=1·7×10−2 respectively). This was further supported by a statistical link (P=5×10−3) between high levels of circulating progesterone and weak bcl‐2 staining in lobules and ducts. This progesterone‐dependent variation was absent in fibroadenomas. No statistical correlation was found between bcl‐2 expression and circulating levels of oestradiol, and follicle‐stimulating or luteotrophic hormones. Although these are only preliminary results, they suggest an influence of progesterone on bcl‐2 expression which might be lost in fibroadenomas. A hypothesis is proposed concerning the potential involvement of altered regulation of the apoptotic process in the formation of such benign lesions.

Strain-dependent variation of the protein composition of Tetrahymena ribosomes

We have described the preparation and preliminary characterization of active ribosomal subunits from the amicronucleate strain CGL of the protozoa Tetrahymerza pyriformis [ 11. Further development of these studies and their extension to micronucleate and to other amicronucleate strains of Tetrahymena has led to revised (higher) estimates of the number of proteins in the subunits of ribosomes of this microorganism and to the observation of large differences between the ribosomal protein complements of different strains. Here we compare the ribosomal proteins of the previously studied amicronucleate strain T. pyriformis CGL and of the micronucleate strain T. thermophila B IV. The revised estimates of the number of proteins in the subunits of ribosomes of T. pyriformis CGL with, in parenthesis, the number reported [ 1 ] are: 40 S subunit; S(2) acidic and 35(3 1) basic proteins 60 S subunit; 6(2/3) acidic and 40(35) basic proteins Ribosomal subunits of T. thermophila B IV have the following protein contents: 40 S subunit; 4 acidic and 34 basic proteins 60 S subunit; 5 acidic and 38 basic proteins Comparison of the electrophoretic properties of the basic ribosomal proteins of T. pyriformis CGL and T. thermophila B IV in Wittmann 2D system shows

Monovalent cation-dependent reversible phosphorylation of a 40 S ribosomal subunit protein in growth-arrested Tetrahymena: correlation with changes in intracellular pH

Phosphorylation and dephosphorylation of ribosomal protein S8 in starved Tetrahymena induced respectively by Na+ and K+ are accompanied by changes in intracellular pH which rises by about 0.8 pH units in cells starving in the presence of Na+ (phosphorylation of S8) and falls by a little more than one pH unit after subsequent addition of K+ (dephosphorylation of S8).