The antiproliferative action of the retinoblastoma tumor suppressor protein, RB, is disrupted in the majority of human cancers. Disruption of RB activity occurs through several disparate mechanisms, including viral oncoprotein binding, deregulated RB phosphorylation, and mutation of the RB gene. Here we report disruption of RB-signaling in tumor cells through loss of a critical cooperating factor. We have previously reported that C33A cells fail to undergo cell cycle inhibition in the presence of constitutively active RB (PSM-RB). To determine how C33A cells evade RB-mediated arrest, cell fusion experiments were performed with RB-sensitive cells. The resulting fusions were arrested by PSM-RB, indicating that C33A cells lack a factor required for RB-mediated cell cycle inhibition. C33A cells are deficient in BRG-1, a SWI͞SNF family member known to stimulate RB activity. Consistent with BRG-1 deficiency underlying resistance to RB-mediated arrest, we identified two other BRG-1-deficient cell lines (SW13 and PANC-1) and demonstrate that these tumor lines are also resistant to cell cycle inhibition by PSM-RB and p16ink4a, which activates endogenous RB. In cell lines lacking BRG-1, we noted a profound defect in RB-mediated repression of the cyclin A promoter. This deficiency in RB-mediated transcriptional repression and cell cycle inhibition was rescued through ectopic coexpression of BRG-1. We also demonstrate that 3T3-derived cells, which inducibly express a dominant-negative BRG-1, arrest by PSM-RB and p16ink4a in the absence of dominant-negative BRG-1 expression; however, cell cycle arrest was abrogated on induction of dominant-negative BRG-1. These findings demonstrate that BRG-1 loss renders cells resistant to RB-mediated cell cycle progression, and that disruption of RB signaling through loss of cooperating factors occurs in cancer cells.cyclins ͉ Cdk ͉ SWI͞SNF T he retinoblastoma tumor suppressor protein (RB) is a critical regulator of cell cycle progression that is functionally inactivated in the majority of human tumors (1-8). RB functions as a protein-binding protein, binding to greater than 50 identified cellular proteins. However, the requirement of these proteins for RB-mediated cell cycle inhibition is largely unknown. Overall, RB-assembled protein complexes lead to the repression of transcription, and this function of RB is critical for cell cycle regulation. The principal target of RB is believed to be the E2F family of transcriptional activators (6, 9-11). E2F controls the expression of numerous genes directly involved in cell cycle progression or in metabolic processes coupled to DNA replication (6, 9-11). RB binding converts E2F from a transcriptional activator to a repressor through a mechanism that involves the recruitment of histone deacetylases (12, 13). RB also mediates the repression of other gene products, such as cyclin A, through complicated mechanisms that are not clearly understood (14).In response to mitogenic signaling, RB is phosphorylated in mid-G 1 by Cdk4͞cyclin D complexes (1-4)....

Aberrant regulation of CD44, a transmembrane glycoprotein, has been implicated in the growth and metastasis of numerous tumors. Although both CD44 overexpression and loss have been implicated in tumor progression, the mechanism of CD44 down-regulation in these tumor types is not known. By immunoblot and reverse transcription-polymerase chain reaction analysis we determined that a cervical carcinoma cell line, C33A, lacks CD44 expression. To determine how CD44 is down-regulated in C33A cells, we utilized cell fusions of C33A cells with a CD44-expressing cell line (SAOS-2). We found that SAOS-2 fusion restored CD44 expression in C33A cells, suggesting that a trans-acting factor present in SAOS-2 cells promotes CD44 production. C33A cells are BRG-1-deficient, and we found that CD44 was absent in another BRG-1-deficient tumor cell line, indicating that loss of BRG-1 may be a general mechanism by which cells lose CD44. Reintroduction of BRG-1 into these cells restored CD44 expression. Furthermore, disruption of BRG-1 function through the use of dominant-negative BRG-1 demonstrated the requirement of BRG-1 in CD44 regulation. Finally, we show that Cyclin E overexpression resulted in the attenuation of CD44 stimulation, which is consistent with previous observations that Cyclin E can abrogate BRG-1 action. Taken together, these results suggest that BRG-1 is a critical regulator of CD44 expression, thus implicating SWI/SNF components in the regulation of cellular adhesion and metastasis.The CD44 family of transmembrane glycoproteins has been implicated in cell-cell and cell-matrix adhesion, cell growth, and metastasis (1-3). A number of different CD44 proteins are produced through alternative RNA splicing, and these proteins are extensively modified. Many tumors express higher than normal levels of total CD44 protein as well as splice variants that do not occur in normal cells (1,3, 4). How CD44 expression is regulated in normal cells and in tumors is poorly understood.A role for CD44 in tumor progression has been documented in numerous clinical and experimental studies (1, 2). Ectopic expression of some forms of CD44 can enhance metastasis and tumor growth both in vitro and in vivo (5-7). It is believed that CD44 expression in some tumors increases as the tumor becomes more proliferative and invasive (1). These findings suggest that CD44 might be regulated by environmental or genetic factors that have been shown to contribute to cancer progression. The expression of activated oncogenes like v-Ras, v-Src, and v-Fos, which promote transformation and invasion, have been reported to induce CD44 expression (8 -10). In addition, the epidermal growth factor receptor has also been shown to stimulate CD44 (10,11).In contrast to studies that correlate CD44 overexpression with cancer progression, a significant number of reports also indicate that loss of CD44 expression can contribute to tumorigenesis (12). Specifically, it has been shown that loss of CD44 in cervical carcinomas, neuroblastomas, prostate carcinomas, melanomas, an...

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Sacubitril/valsartan (LCZ696) is the first angiotensin receptor neprilysin inhibitor approved to reduce cardiovascular mortality and hospitalization in patients with heart failure with reduced ejection fraction. As neprilysin (NEP) is one of several enzymes known to degrade amyloid-β (Aβ), there is a theoretical risk of Aβ accumulation following long-term NEP inhibition. The primary objective of this study was to evaluate the potential effects of sacubitril/valsartan on central nervous system clearance of Aβ isoforms in cynomolgus monkeys using the sensitive Stable Isotope Labeling Kinetics (SILK™)-Aβ methodology. The in vitro selectivity of valsartan, sacubitril, and its active metabolite sacubitrilat was established; sacubitrilat did not inhibit other human Aβ-degrading metalloproteases. In a 2-week study, sacubitril/valsartan (50mg/kg/day) or vehicle was orally administered to female cynomolgus monkeys in conjunction with SILK™-Aβ. Despite low cerebrospinal fluid (CSF) and brain penetration, CSF exposure to sacubitril was sufficient to inhibit NEP and resulted in an increase in the elimination half-life of Aβ1-42 (65.3%; p=0.026), Aβ1-40 (35.2%; p=0.04) and Aβtotal (29.8%; p=0.04) acutely; this returned to normal as expected with repeated dosing for 15days. CSF concentrations of newly generated Aβ (AUC) indicated elevations in the more aggregable form Aβ1-42 on day 1 (20.4%; p=0.039) and day 15 (34.7%; p=0.0003) and in shorter forms Aβ1-40 (23.4%; p=0.009), Aβ1-38 (64.1%; p=0.0001) and Aβtotal (50.45%; p=0.00002) on day 15. However, there were no elevations in any Aβ isoforms in the brains of these monkeys on day 16. In a second study cynomolgus monkeys were administered sacubitril/valsartan (300mg/kg) or vehicle control for 39weeks; no microscopic brain changes or Aβ deposition, as assessed by immunohistochemical staining, were present. Further clinical studies are planned to address the relevance of these findings.

The observed effects in juvenile rats were considered predictable and primarily related to the mechanism of action of letrozole upon estrogen synthesis.