Binding of the aminothiol WR-1065 to transcription factors influences cellular response to anticancer drugs

ABSTRACT The aminothiol WR-1065 (the active form of amifostine) protects normal tissues from the toxic effects of certain cancer drugs, while leaving their antitumor effects unchanged. The present data address the mechanism of action of this dichotomous effect. 35 S-Labeled WR-1065 bound directly to the transcription factors nuclear factor-B, activator protein-1, and p53, resulting in enhanced binding of these proteins to target regulatory DNA sequences and subsequent transactivation of a number of downstream genes. Since other small molecular thiols could mimic WR-1065, the redox potential of the sulfhydryl is an important determinant of its activity. In nontransformed cells, WR-1065 protected cells from the cytotoxic effects of paclitaxel in a p53-dependent manner. However, in a transformed human tumor cell line, there was no cytoprotectivity by WR-1065, consistent with the premise that p53-dependent growth arrest is the basis for the protective effect of this compound, and that this pathway is abrogated in human tumors. The combined data support the principle that the cellular effects of the aminothiol WR-1065 are mediated through an impact on transcriptional regulation and are not only a consequence of radical scavenging

The tuberous sclerosis proteins regulate formation of the primary cilium via a rapamycin-insensitive and polycystin 1-independent pathway

Tuberous sclerosis complex (TSC) is a tumor suppressor gene syndrome in which severe renal cystic disease can occur. Many renal cystic diseases, including autosomal dominant polycystic kidney disease (ADPKD), are associated with absence or dysfunction of the primary cilium. We report here that hamartin (TSC1) localizes to the basal body of the primary cilium, and that Tsc1−/− and Tsc2−/− mouse embryonic fibroblasts (MEFs) are significantly more likely to contain a primary cilium than wild-type controls. In addition, the cilia of Tsc1−/− and Tsc2−/− MEFs are 17–27% longer than cilia from wild-type MEFs. These data suggest a novel type of ciliary disruption in TSC, associated with enhanced cilia development. The TSC1 and TSC2 proteins function as a heterodimer to inhibit the activity of the mammalian target of rapamycin complex 1 (TORC1). The enhanced ciliary formation in the Tsc1−/− and Tsc2−/− MEFs was not abrogated by rapamycin, which indicates a TORC1-independent mechanism. Polycystin 1 (PC1), the product of the PKD1 gene, has been found to interact with TSC2, but Pkd1−/− MEFs did not have enhanced ciliary formation. Furthermore, while activation of mTOR has been observed in renal cysts from ADPKD patients, Pkd1−/− MEFs did not have evidence of constitutive mTOR activation, thereby underscoring the independent functions of the TSC proteins and PC1 in regulation of primary cilia and mTOR. Our data link the TSC proteins with the primary cilium and reveal a novel phenotype of enhanced ciliary formation in a cyst-associated disease

Tumor Suppressive Functions of p53

The majority of human cancers acquire mutations that abrogate the p53 tumor suppressor network and, as a consequence, p53 is one of the most extensively studied proteins in cancer research. Because of its potent tumor suppressive activity, it is widely assumed that a molecular understanding of p53 action will produce fundamental insights into natural processes that limit tumorigenesis and may identify key molecular targets for therapeutic intervention. p53 functions largely as a transcription factor, and can trigger a variety of antiproliferative programs by activating or repressing key effector genes. Despite a significant body of literature detailing the biochemical and biological functions of p53, much remains to be elucidated. Indeed, the p53 network is as complex and enigmatic as it is relevant. It is the goal of this article, written 30 years after the discovery of p53, to present a concise review of the tumor suppressor role of the p53 network and to highlight the context-dependent nature of p53 target-gene functions

Suppression of tumorigenesis by the p53 target PUMA

The p53 tumor suppressor regulates diverse antiproliferative processes such that cells acquiring p53 mutations have impaired cell-cycle checkpoints, senescence, apoptosis, and genomic stability. Here, we use stable RNA interference to examine the role of PUMA, a p53 target gene and proapoptotic member of the Bcl2 family, in p53-mediated tumor suppression. PUMA short hairpin RNAs (shRNAs) efficiently suppressed PUMA expression and p53-dependent apoptosis but did not impair nonapoptotic functions of p53. Like p53 shRNAs, PUMA shRNAs promoted oncogenic transformation of primary murine fibroblasts by the E1A/ras oncogene combination and dramatically accelerated myc-induced lymphomagenesis without disrupting p53-dependent cell-cycle arrest. However, the ability of PUMA to execute p53 tumor suppressor functions was variable because, in contrast to p53 shRNAs, PUMA shRNAs were unable to cooperate with oncogenic ras in transformation. These results demonstrate that the p53 effector functions involved in tumor suppression are context dependent and, in some settings, depend heavily on the expression of a single proapoptotic effector. Additionally, they demonstrate the utility of RNA interference for evaluating putative tumor suppressor genes in vivo

The retinoblastoma tumor suppressor modifies the therapeutic response of breast cancer

The retinoblastoma tumor suppressor (RB) protein is functionally inactivated in the majority of human cancers and is aberrant in one-third of all breast cancers. RB regulates G(1)/S-phase cell-cycle progression and is a critical mediator of antiproliferative signaling. Here the specific impact of RB deficiency on E2F-regulated gene expression, tumorigenic proliferation, and the response to 2 distinct lines of therapy was investigated in breast cancer cells. RB knockdown resulted in RB/E2F target gene deregulation and accelerated tumorigenic proliferation, thereby demonstrating that even in the context of a complex tumor cell genome, RB status exerts significant control over proliferation. Furthermore, the RB deficiency compromised the short-term cell-cycle inhibition following cisplatin, ionizing radiation, and antiestrogen therapy. In the context of DNA-damaging agents, this bypass resulted in increased sensitivity to these agents in cell culture and xenograft models. In contrast, the bypass of antiestrogen signaling resulted in continued proliferation and xenograft tumor growth in the presence of tamoxifen. These effects of aberrations in RB function were recapitulated by ectopic E2F expression, indicating that control of downstream target genes was an important determinant of the observed responses. Specific analyses of an RB gene expression signature in 60 human patients indicated that deregulation of this pathway was associated with early recurrence following tamoxifen monotherapy. Thus, because the RB pathway is a critical determinant of tumorigenic proliferation and differential therapeutic response, it may represent a critical basis for directing therapy in the treatment of breast cancer

An epi-allelic series of p53 hypomorphs created by stable RNAi produces distinct tumor phenotypes in vivo

The application of RNA interference (RNAi) to mammalian systems has the potential to revolutionize genetics and produce novel therapies. Here we investigate whether RNAi applied to a well-characterized gene can stably suppress gene expression in hematopoietic stem cells and produce detectable phenotypes in mice. Deletion of the Trp53 tumor suppressor gene greatly accelerates Myc-induced lymphomagenesis, resulting in highly disseminated disease(1,2). To determine whether RNAi suppression of Trp53 could produce a similar phenotype, we introduced several Trp53 short hairpin RNAs (shRNAs) into hematopoietic stem cells derived from Emu-Myc transgenic mice, and monitored tumor onset and overall pathology in lethally irradiated recipients. Different Trp53 shRNAs produced distinct phenotypes in vivo, ranging from benign lymphoid hyperplasias to highly disseminated lymphomas that paralleled Trp53(-/-) lymphomagenesis in the Emu-Myc mouse. In all cases, the severity and type of disease correlated with the extent to which specific shRNAs inhibited p53 activity. Therefore, RNAi can stably suppress gene expression in stem cells and reconstituted organs derived from those cells. In addition, intrinsic differences between individual shRNA expression vectors targeting the same gene can be used to create an 'epi-allelic series' for dissecting gene function in vivo