Estrogen receptor-A (ER-A) plays a crucial role in normal breast development and has also been linked to mammary carcinogenesis and clinical outcome in breast cancer patients. However, ER-a gene expression can change during the course of disease and, consequently, therapy resistance can occur. The molecular mechanism governing ER-a transcriptional activity and/or silencing is still unclear. Here, we showed that the presence of a specific pRb2/p130 multimolecular complex on the ER-a promoter strongly correlates with the methylation status of this gene. Furthermore, we suggested that pRb2/ p130 could cooperate with ICBP90 (inverted CCAAT box binding protein of 90 kDa) and DNA methyltransferases in maintaining a specific methylation pattern of ER-a gene. The sequence of epigenetic events for establishing and maintaining the silenced state of ER-a gene can be locus-or pathwayspecific, and the local remodeling of ER-a chromatin structure by pRb2/p130 multimolecular complexes may influence its susceptibility to specific DNA methylation. Our novel hypothesis could provide a basis for understanding how the complex pattern of ER-a methylation and transcriptional silencing is generated and for understanding the relationship between this pattern and its function during the neoplastic process. [Cancer Res 2007;67(16):7731-7]

The putative role of TP53 and p16 INK4A tumor suppressor genes and Ras oncogenes in the development and progression of salivary gland neoplasias was studied in 28 cases of pleomorphic adenomas (PA), 4 cases of cystic adenocarcinomas, and 1 case of carcinoma ex-PA. Genetic and epigenetic alterations in the above genes were analyzed by Polymerase Chain Reaction/Single Strand Conformational Polymorphism (PCR/SSCP) and sequencing and by Methylation Specific-PCR (MS-PCR). Mutations in TP53 were found in 14% (4/28) of PAs and in 60% (3/5) of carcinomas. Mutations in H-Ras and K-Ras were identified in 4% (1/28) and 7% (2/28) of PAs, respectively. Only 20% (1/5) of carcinomas screened displayed mutations in K-Ras. p16 INK4A promoter hypermethylation was found in 14% (4/28) of PAs and 100% (5/5) carcinomas. All genetic and epigenetic alterations were detected exclusively in the epithelial and transitional tumor components, and were absent in the mesenchymal parts. Our analysis suggests that TP53 mutations and p16 INK4A promoter methylation, but not alterations in the H-Ras and K-Ras genes, might be involved in the malignant progression of PA into carcinoma.

Genotype analysis is becoming more and more useful in clinical practice, since specific mutations in tumors often correlate with prognosis and/or therapeutic response. Unfortunately, current molecular analytical techniques often require time-consuming and costly steps of analysis, thus making their routine clinical use difficult. Moreover, one of the most difficult problems arising during tumor research is that of their cell heterogeneity, which depends on their clear molecular heterogeneity. SSCP analysis discriminates by means of aberrant electrophoresis migration bands, mutated alleles which may represent as little as 15-20% of their total number. Nevertheless, in order to identify by sequencing the type of alteration revealed by this technique, only the mutated allele must be isolated. The advent of laser microdissection is a procedure which easily solves these problems of accuracy, costs, and time. The aims of this study were to perfect the system of laser pressure catapulting (LPC) laser microdissection for the assessment of the mutational status of p53 and k-ras genes in a consecutive series of 67 patients with colorectal carcinomas (CRC), in order to compare this technique with that involving hand-dissection and to demonstrate that since the LPC system guarantees more accurate biomolecular analyses, it should become part of clinical routine in this field. The LPC-system was perfected with the use of mineral oil and the LPCmembrane. To compare the techniques of hand-and LPC-microdissection, alcohol-fixed, paraffin-embedded tissue from 67 cases of CRC were both handand laser-microdissected. In either case, dissected samples were analyzed by SSCP/ sequencing and direct sequencing for k-ras and p53 gene mutations. LPCmicrodissection made it possible to pick up mutations by direct sequencing or SSCP/sequencing, whereas hand-microdissection mutations were identified only by means of SSCP followed by sequencing; direct sequencing did not reveal any mutation. In the 67 patients examined by either method, 36% (24/67) showed p53 mutations, 32 of which identified. Seventy-eight percent (25/32) were found in the conserved areas of the gene, while 12% (4/32) were in the L2 loop, 50% (16/32) were in the L3 loop, and 12% (4/32) in the LSH motif of the protein. Moreover, of the 67 cases examined, 40% (27/67) showed mutations in k-ras, with a total of 29 mutations identified. Of these, 14 (48%) were found in codon 12 and 15 (52%) in codon 13. The modifications which we brought to the LPC system led to a vast improvement of the technique, making it an ideal substitution for handmicrodissection and guaranteeing a considerable number of advantages regarding facility, accuracy, time, and cost. Furthermore, the data obtained from the ß 2004 WILEY-LISS, INC.The authorship is shared equally by Viviana Bazan and Gaspare La Rocca.

Estrogens exhibit important biological functions and influence several pathological processes of hormone-dependent diseases. The biological actions of estrogens require their interaction with two estrogen receptors (ER-alpha and ER-beta), which are ligand-dependent transcription factors. ER-alpha and ER-beta exhibit distinct tissue expression patterns as well as show different patterns of gene regulation. In addition, it has been suggested that ER-beta works as a counter partner of ER-alpha through inhibition of the transactivating functions of ER-alpha. For instance, ER-beta seems to play a different role in breast tumorigenesis than ER-alpha, as ER-beta decreased expression in breast cancer has been correlated with bad prognosis. Biological activities of ER-alpha and ER-beta could be controlled by a number of interacting proteins such as activators/inhibitors, ligand binding and kinases. We have previously reported that pRb2/p130, retinoblastoma related protein, could be involved in the silencing of ER-alpha gene during breast tumorigenesis. Here, we report that ER-beta and pRb2/p130 proteins co-immunoprecipitate in both nucleus and cytoplasm of MCF-7 breast cancer cells. Our hypothesis is that the interaction of pRb2/130 with ER-beta may have a functional significance in regulating ER-beta activity.