Differential Expression of matrix Attachment Region-Binding Proteins in Human Prostatic cancer cell lines

Background: Abnormal nuclear organization and alterations in the amount and distribution of heterochromatin have always been recognized as hallmarks of human cancer. However, neither the exact cause of these modifications nor how the activity/silencing of thousands genes can be orchestrated are currently known. In eukaryotes, the genome is compartmentalized into chromatin domains by attachment to a supporting structuretermed the nuclear matrix (NM). The interactions of chromatin with the NM occur via AT-rich DNA sequences called matrix attachment regions (MARs). MARs exibit several functions including the organization of chromatin loops, the augmentation of transcription and the facilitation of replication; moreover, the attachment of the genome to the NM is a dynamic event that is cell type or cell cycle-dependent. Several MAR-binding proteins have been identified, some are drammatically dysregulated in tumor cells and their expression is often significantly correlated with more aggressive phenotype. Likewise, modifications of the interactions between NM preoteins and MARs might be related to the large-scale chromatin organization observed in carcinogenesis. research carried out in our and other laboratories seeking prostate cancer (PCa) markers with improved diagnostic and prognostic features has identified some NMproteins that are differentially expressed in PCa with respect to non-tumor tissue; a few of these proteins were significantly correlated with tumor aggressiveness and/or risk of biochemical progression. These findings prompted us to characterize the changes in the NM protein-MAR bond in PCa. Materials and methods: in this study, using a proteomic approach along with two-dimensional Southwestern analysis, we charaterized the NM protein-MAR bond in two prostatic cancer cell lines: the androgen-responsive LNCaP and the androgen-resistant PC3. For binding experiments, a highly repetitive bent DNA sequence of 370-bp (XmnI) containing a base unpairing region was used as a probe. The variation of the expression of some MAR proteins was determined by quantitative Western blot. Both the nucleoplasm distribution and the co-localization with DNA were evaluated by confocal laser scanning microscopy. Using transmission electron microscopy (TEM) the high-order structure of chromatin was examined in the two cell lines. Results: several NM proteins extracted from both cell lines strongly bind to the XmnI sequence. It is possible to separate these proteins into two groups: I. MAR-binding proteins present in a large quantity also in other cellular types (MARPa, MARPb, hnRNPs, Lamin A, Lamin B, Matrin3) that may correspond to the proteins in contact with the constitutive MARs (i.e. the sequences demarcating permanent domain boundaries in all cell types). II. MAR-binding proteins corresponding to cell-type specific factors (PARPs, SATB1) that interact with facultative MARs (i.e. the sequences cell type- and activity- related and dependent on cell differentiation). In PC3 with respect to LNCaP cells, several changes in the ability of proteins to bind XmnI sequence were observed: the signal intensity of Matrin3, SATB1 and all basic hnRNPs decreased, showing that these proteins bind the DNA more weakly or are down-regulated in more aggressive cells. In contrast, MARPa, MARPb, PARP and its fragments increased. Confocal microscopy demonstrated that also the co-localization of PARP with DNA was significantly lower. These changes were concomitant with a different compartmentalization of chromatin in the two cell lines, as shown by TEM. Conclusion: our study provides evidence that several MAR-binding proteins undergo extensive changes in PC3 with respect to LNCaP cells, showing that the interactions between NM and the base of the chromatin loop are involved in cell differentiation. These proteins may turn out to be an important tool in the understanding of PCa carcinogenesis and may be novel targets of anticancer drugs.