Nuclear hormone receptor NR4A2 is involved in cell transformation and apoptosis.

HeLaHF cells are transformation revertants of cervical cancer HeLa cells and have lost anchorage-independent growth potential and tumorigenicity. Activation of tumor suppressor(s) was implicated previously in this transformation reversion. In this study, expression profiling analysis was carried out to identify potential oncogenes that are down-regulated in HeLaHF cells. We found that all three members of the NR4A1/Nur77/NGFIB orphan nuclear hormone receptor subfamily (NR4A1, NR4A2, and NR4A3) were down-regulated in the HeLaHF revertant. Small interfering RNA-mediated down-regulation of NR4A2 in HeLa cells, either transiently or stably, resulted in reduced anchorage-independent growth that was largely attributable to increased anoikis. Furthermore, down-regulation of NR4A2 as well as NR4A1 promoted intrinsic apoptosis. These phenotypes were also observed in several other experimental cancer cells, suggesting the observed apoptosis suppression is a more general property of NR4A2 and NR4A1. These phenotypes also suggest that the Nur77/NGFIB subfamily of orphan receptors exhibit certain oncogenic functionalities with regards to cell proliferation and apoptosis and could therefore be evaluated as potential cancer therapeutic targets.

A 96-well surrogate survival assay coupled with a special short interfering RNA vector for assessing cancer gene targets with enhanced signal/noise ratio and its utility in HTS for cancer therapeutic targets.

Transient transfection of short interfering RNAs to inactivate cancer therapeutic genes in cancer cells is an important method to induce therapeutic phenotypes (cell apoptosis, growth arrest, etc.) for cancer target validation. These phenotypes can be initially assessed by cell survival via colorimetric/fluorescence readings, e.g., alamarBlue (Trek Diagnostic Systems, Cleveland, OH) and WST-1. However, intrinsic problems exist for transient transfection-varying toxicity, inconsistent transfection efficiency, as well as other cell-specific determinants-which contribute to a low signal:noise ratio of the assays, rendering of the assay ineffective particularly when applied in high-throughput screening (HTS) multiplexed for different cells. This report describes a method using reporter as a "normalized surrogate" for the conventional survival readout in a 96-well format. In this approach, only the transfected surviving cells produce reporter activities, and many variables associated with transient transfection are excluded. A constitutively expressed reporter gene (luciferase or LacZ) expression cassette is co-transfected into cells along with a specially designed RNA interference (RNAi) vector (or a transgene for that matter). The reporter activity in either liquid cultures or in soft agar cultures in 96-well formats is then quantitated in situ. The RNAi vector construction is simplified so that it can be adapted to a 96-well format. Our data demonstrated that the relative reporter readings for survival are independent of both transfection efficiency and cellular toxicity. The signal:noise ratio is markedly increased, particularly for cells with low transfection efficiency. The assay is versatile and robust and can be applied in multiplexed HTS for cancer target identification and validation.

MDDD, a 4,9-diazapyrenium derivative, is selectively toxic to glioma cells by inducing growth arrest at G0/G1 independently of p53.

4-Methyl-2,7-diamino-5,10-diphenyl-4,9-diaz-apyrenium chloride (MDDD), a stable and water soluble nucleic acid-intercalating agent, was shown to be toxic to cancer cells with IC50 around 10 microM. IC(50) We tested MDDD for its potential antitumor activities and found it inhibited cancer cell growth with IC(50) in the micromolar range for the majority of cancer cells tested, with the exception of glioma cells, for which the IC(50) is in the submicromolar range. This unique selectivity of MDDD to glioma cells can potentially be exploited for anti-glioma therapeutics. Although the underlying mechanisms for the apparent glioma specificity remain to be elucidated, our analysis indicates that MDDD significantly reduces cell clonogenicity and blockes cell proliferation at the G1 phase. MDDD treatment also triggers induction of p53 and p21 at the protein levels, suggesting the activation of DNA damage response. However, MDDD mediated growth inhibition does not require the p53 pathway since p53+/- isogenic cell pairs display the same sensitivity. These properties of MDDD favor its candidacy for evaluation as a new anti-tumor agent, particularly for glioma.

PHTS, a novel putative tumor suppressor, is involved in the transformation reversion of HeLaHF cells independently of the p53 pathway.

HeLaHF is a non-transformed revertant of HeLa cells, likely resulting from the activation of a putative tumor suppressor(s). p53 protein was stabilized in this revertant and reactivated for certain transactivation functions. Although p53 stabilization has not conclusively been linked to the reversion, it is clear that the genes in p53 pathway are involved. The present study confirms the direct role of p53 in HeLaHF reversion by demonstrating that RNAi-mediated p53 silencing partially restores anchorage-independent growth potential of the revertant through the suppression of anoikis. In addition, we identified a novel gene, named PHTS, with putative tumor suppressor properties, and showed that this gene is also involved in HeLaHF reversion independently of the p53 pathway. Expression profiling revealed that PHTS is one of the genes that is up-regulated in HeLaHF but not in HeLa. It encodes a putative protein with CD59-like domains. RNAi-mediated PHTS silencing resulted in the partial restoration of transformation (anchorage-independent growth) in HeLaHF cells, similar to that of p53 gene silencing, implying its tumor suppressor effect. However, the observed increased transformation potential by PHTS silencing appears to be due to an increased anchorage-independent proliferation rate rather than suppression of anoikis, unlike the effect of p53 silencing. p53 silencing did not affect PHTS gene expression, and vice versa, suggesting PHTS may function in a new and p53-independent tumor suppressor pathway. Furthermore, over-expression of PHTS in different cancer cell lines, in addition to HeLa, reduces cell growth likely via induced apoptosis, confirming the broad PHTS tumor suppressor properties.