Change in the expression of a nuclear matrix-associated protein is correlated with cellular transformation.

Abstract

We have identified a monoclonal antibody that recognizes a nuclear matrix-associated protein in NIH 3T3 cells. The immunofluorescence pattern consists predominantly of bright nuclear granule clusters distributed throughout the nucleoplasm, with the exclusion of nucleoli. It recognizes a protein of 190 kDa that is down-regulated to various degrees in a panel of single-oncogene-transformed NIH 3T3 cells. Its localization is similar, but not identical, to the spliceosomal speckles. p190 shows a coordinate expression during the growth cycle of nontransformed NIH 3T3 cells; it is synthesized at the highest level under growth arrest conditions. It is expressed in adult mouse brain and is also present in human IMR-90 fibroblasts. Cellular transformation is accompanied by gross changes in the architecture ofboth nuclear and cytoplasmic organization (1-5). Much data have been accumulated over the past years on the importance that such changes have on transformation parameters, through the integrated knowledge of specific cellular and viral oncogene products. Much less is known about specific changes in the nuclear architecture that relate, directly or indirectly as in the case of cytoskeletal changes, to the transformed phenotype. We For serum starvation, the medium was changed to 0.5% FCS when cells were subconfluent. After 48 hr cells were harvested for analysis. Under these conditions, incubation with 50 gM bromodeoxyuridine (BrdUrd) for an additional 24 hr resulted in labeling of <3% of the nuclei. For induction of DNA synthesis, fresh medium containing 20% FCS was added to the arrested cells; after 24 hr of incubation in the presence of BrdUrd, >90% of the nuclei were labeled. For density-dependent inhibition, cells were plated at 104/cm2 in 10% FCS. Twelve hours after plating (considered as the starting point for growing cells), the medium was changed every 2 days. After 4 days in culture, incubation with BrdUrd for 2 hr resulted in <1% incorporation. Exponentially growing cells correspond to cells that were cultured for 24 hr in 10% FCS. Incubation with BrdUrd for 2 hr after this time resulted in 60o positively stained nuclei. NIH 3T3 transformed with v-ras, v-myc, and v-src (9) were kindly provided by F. Tat6 (University of Rome). NIH 3T3 cells transformed with v-fos were kindly provided by R. Muller.Preparation of mAbs. To prepare the immunogen, NIH 3T3 fibroblasts were washed twice with cold phosphate-buffered saline (PBS). Nuclear/cytoplasmic separation was performed as reported (10). Cell lysis was performed in a buffer containing 10 mM Tris HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, and 0.5% (vol/vol) Nonidet P-40 (NP-40). The homogenate was then centrifuged for 5 min at 500 x g to yield a crude nuclear fraction. The resulting nuclei were extracted in 1% SDS/50 mM Tris, pH 7.5/100 mM NaCl.Eight-week-old BALB/c mice were injected intraperitoneally with a suspension of 0.4 mg of the immunogen in 200 ,ul of complete Freund's adjuvant emulsion (1:1, vol/vol). Two weeks later, an identical injection was perf...

Changes in the expression of cytokeratins and nuclear matrix proteins are correlated with the level of differentiation in human prostate cancer

“…To start the identification of general modulating factors of chromatin superstructure we have recently carried out the protein characterization of the NM-IF complex during rat hepatocyte transformation; we have found that the protein composition undergoes progressive changes synchronous with chromatin decondensation . The new synthesis of NM proteins relating to the emergence of successive cell populations was observed, in line with the contention that a subset of NM proteins is characteristic of the stage of transformation [Pienta et al, 1989;Brancolini and Schneider, 1991]. The actual relationship between chromatin decondensation phenomena and alterations in the composition and/or architecture of the NM still remains to be discovered.…”

The effect of sodium tetrathionate stabilization on the distribution of three nuclear matrix proteins in human K562 erythroleukemia cells

“…Reports from several laboratories have shown that when the matrix is prepared in situ from adherentgrowing cells (Staufenbiel and Deppert 1984) the distinctive fluorescent pattern given by antibodies directed against nuclear antigens which resist extraction with high ionic strength buffers and nuclease digestion is identical to that detected in whole cells (e.g. Brancolini and Schneider 1991;Hirose et al 1988;Kallajoki et al 1991;Philopova et al 1987). These observations have been interpreted as meaning that cells actually contained an inner network acting as a nucleoskeleton.…”

A c-erbB-2 Promoter-specific Nuclear Matrix Protein from Human Breast Tumor Tissues Mediates NF-κB DNA Binding Activity

“…In recent years, intensive studies have also suggested possibly related role(s) of the nuclear matrix in tumor progression (10 -16). In cancer cells, some transforming proteins appear to be associated with the matrix (23,24), and there are also indications of specific alterations in the protein composition of the matrix as cells undergo differentiation (25) and during the invasion and proliferation of tumors (14,15,24,25,42,43). Could a "tumor-specific" NMP be involved in a transition associated with tumor progression?…”