bcl-2 expression is often associated with poor prognosis in several types of tumors; however, the role of this molecule in breast cancer is still controversial. We found earlier that over-expression of bcl-2 in a human breast-cancer cell line (MCF7 ADR ) enhances its tumorigenicity and metastatic potential by inducing metastasis-associated properties such as increased secretion of the matrix metalloproteinase-9 (mmp-9). In the present study, we investigated the effect of bcl-2 over-expression on the activity of the transcription factor NF-B, an important regulator of genes involved in tumor progression and invasion. Transient transfection experiments indicate that over-expression of bcl-2 in the MCF7 ADR cell line, enhances NF-B-dependent transcriptional activity. Mobility-shift analysis revealed an increase of NF-B DNAbinding in bcl-2-over-expressing clones that correlated with lower levels of the NF-B cytoplasmic inhibitor IB␣. Moreover, point mutations of 2 highly conserved residues within the BH1 and BH2 domains that abrogate the interaction of bcl-2 with bax, or deletion of the N-terminal BH4 domain, completely eliminate the ability of this molecule to up-regulate NF-B-dependent transactivation. Since mmp-9 is a NF-B-regulated gene, we also investigated whether bcl-2 overexpression up-regulated mmp-9 transcription. We found that induction of mmp-9 mRNA correlates with the activation of an mmp-9-promoter-reporter-gene construct in transient transfection assay, and a mutation of the (؊600)mmp-9-NF-B binding element abolishes this effect. The overall data indicate that bcl-2-mediated regulation of NF-B-transcription-factor activity may represent an important mechanism for the promotion of malignant behavior in MCF-7 ADR cells.

bcl‐2 expression is often associated with poor prognosis in several types of tumors; however, the role of this molecule in breast cancer is still controversial. We found earlier that over‐expression of bcl‐2 in a human breast‐cancer cell line (MCF7ADR) enhances its tumorigenicity and metastatic potential by inducing metastasis‐associated properties such as increased secretion of the matrix metalloproteinase‐9 (mmp‐9). In the present study, we investigated the effect of bcl‐2 over‐expression on the activity of the transcription factor NF‐κB, an important regulator of genes involved in tumor progression and invasion. Transient transfection experiments indicate that over‐expression of bcl‐2 in the MCF7ADR cell line, enhances NF‐κB‐dependent transcriptional activity. Mobility‐shift analysis revealed an increase of NF‐κB DNA‐binding in bcl‐2‐over‐expressing clones that correlated with lower levels of the NF‐κB cytoplasmic inhibitor IκBα. Moreover, point mutations of 2 highly conserved residues within the BH1 and BH2 domains that abrogate the interaction of bcl‐2 with bax, or deletion of the N‐terminal BH4 domain, completely eliminate the ability of this molecule to up‐regulate NF‐κB‐dependent transactivation. Since mmp‐9 is a NF‐κB‐regulated gene, we also investigated whether bcl‐2 over‐expression up‐regulated mmp‐9 transcription. We found that induction of mmp‐9 mRNA correlates with the activation of an mmp‐9‐promoter‐reporter‐gene construct in transient transfection assay, and a mutation of the (−600)mmp‐9‐NF‐κB binding element abolishes this effect. The overall data indicate that bcl‐2‐mediated regulation of NF‐κB‐transcription‐factor activity may represent an important mechanism for the promotion of malignant behavior in MCF‐7ADR cells. Int. J. Cancer 86:188–196, 2000. © 2000 Wiley‐Liss, Inc.

The mammalian nuclear factor-kappa B (NF-kB) family comprises 5 members (p50, p52, relA/p65, c-rel and relB) which form a different array of homo-and hetero-dimers and are regulated by a group of citoplasmic inhibitors (IBs) (Krappmann et al, 1999). In unstimulated cells, it is retained in an inactive cytoplasmatic form by one of several IB molecules. Activation of NF-kB complexes by a variety of stimuli results in the phosphorylation and rapid degradation of IBs through the ubiquitin-proteasome pathway (Thanos and Maniatis, 1999). Dissociation of IB from NF-kB allows NF-kB to translocate to the nucleus and bind to B DNA sites with the consequent activation of B-target gene expression (Baeuerle and Henkel, 1994).The rel/NF-kB/IB superfamily of signal transducers and transcription factors exists in virtually all cell types but has been characterized as a mediator of response to several stimuli in the immune system (Baeuerle and Henkel, 1994;Krappmann et al, 1999;Thanos and Maniatis, 1999). More recently, it has been shown that NF-kB can also regulate diverse cellular processes such as apoptosis (Abbadie et al, 1993;Lin et al, 1995;Carter et al, 1996;Grilli et al, 1996;Grimm et al, 1996;Wang et al, 1998;Wu et al, 1998;Pahl, 1999;Lin et al, 1999;Grilli and Memo, 1999;Kaltschmidt et al, 2000), cell cycle (Baeuerle and Baltimore, 1996;Bargou et al, 1997;Bash et al, 1997;Seitz et al, 1998;Sheehy and Schlissel, 1999) and oncogenesis (Higgins et al, 1993;Gilmore, 1997;Nakshatri et al, 1997;Sovak et al, 1997;Visconti et al, 1997;Rayet and Gélinas, 1999;Andela et al, 2000;Huang et al, 2000).A variety of studies encompassing a broad range of both cell types and apoptosis-inducing stimuli have evidenced that activation of NF-kB plays a role in both protecting and inducing apoptosis. In a number of systems, NF-kB has been demonstrated to have an antiapoptotic function, and several NF-kB responsive anti-apoptotic genes (clAP1, clAP2, TRAF1, TRAF2, IEXL-1) have recently been identified and claimed to play a role in this process (Wang et al, 1998;Wu et al, 1998;Pahl, 1999). Activation of NF-kB has also been correlated with the activation of the apoptotic programme in a wide variety of systems such as avian embryonic development, ceramide-activated osteoblasts, dopaminergic neurons derived from Parkinson disease patients, bone marrow cells and prostate carcinoma cells (Abbadie et al, 1993;Lin et al, 1995Lin et al, ,1999Carter et al, 1996;Grilli et al, 1996;Grimm et al, 1996). The pro-and antiapoptotic regulatory function of NF-kB has been shown to depend on the cell type, the differentiation state of the cell, and the nature of the apoptotic stimulus (Abbadie et al, 1993;Lin et al, 1999;Kaltschmidt et al, 2000). Given such a divergent outcome, it has been suggested that NF-kB activation acts as a checkpoint between cell rescue and apoptosis (Grilli and Memo, 1999).As reported for the NF-kB role on apoptosis, a different effect on cell proliferation has also been demonstrated. In fact, in contrast to its role in HeLa cells and in...

In this article, we investigated the effect induced by the ify the cell cycle profile in respect of Ad-p53 infected cells. reintroduction of wild-type p53 (wt-p53) protein on BCNU In contrast, BCNU→Ad-p53 sequence provoked G2-M sensitivity in the ADF glioblastoma line. Using a wt-p53 rearrest similar to that observed after treatment with BCNU combinant adenovirus (Ad-p53), we demonstrated that alone, but prevented the later recovery of the cells through exogenous wt-p53 expression was able to increase the the cell cycle, by driving the cells to apoptotic death. These sensitivity to BCNU in ADF cells. Interestingly, this effect results demonstrate that the administration sequence is was more evident when Ad-p53 infection was performed important to increase drug sensitivity. To generalize the after BCNU treatment compared with the opposite phenomenon observed on ADF line, the antiproliferative sequence. To understand the biological basis of these difeffect of the two different schedules was analyzed on other ferent behaviors, we analyzed the cell cycle of the differglioblastoma lines (A172, CRS-A2, U373MG) with different ently treated cells. We found that Ad-p53 infection induced BCNU sensitivity and p53 status. The data obtained a persistent accumulation of cells in the G0/G1 phase confirm that the wt-p53 gene transfer enhances BCNU while, as expected, BCNU induced a block in the G2-M sensitivity in glioblastoma cells depending on the phase. Ad-p53→BCNU sequence did not significantly modadministration sequence.

c-myb antisense oligonucleotides (AS ODNs) were reversibly immobilized to a novel polymeric core shell nanosphere and their cellular uptake and inhibitory effect on HL60 leukemia cell proliferation studied. The nanosphere surface was so designed as to directly bind ODNs via ionic interactions and reversibly release them inside the cells. Compared with the cellular uptake of free oligonucleotide, the use of AS ODN (immobilized to the nanospheres) produced a 50-fold increase in the intracellular concentration. Specifically, a single dose of 320 nM of AS ODN immobilized to the nanospheres was capable of inhibiting HL60 cell proliferation with the same degree of efficiency obtained using a 50-fold higher dose of free AS ODN. Flow cytometric experiments with fluoresceinated ODNs showed a temperature-dependent uptake, which was detectable as early as 2 h after the beginning of treatment. The inhibitory effect on cell proliferation was maintained for up to 8 days of culture. Moreover, the level of c-Myb protein decreased by 24% after 2 days and by 60% after 4 days of treatment, thus indicating a continuous and sustained release of non-degraded AS ODN from the nanospheres inside the cells.