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Netrin-1 may promote colorectal and breast tumorigenesis, by inhibiting apoptosis induced by its dependence receptors, deleted in colorectal cancer (DCC) and uncoordinated-5-homolog (UNC5H). The status of netrin-1 and its receptors in non–small cell lung cancer (NSCLC) was unknown. The levels of netrin-1 and its receptors were analyzed in a panel of 92 NSCLC and 25 human lung cancer cell lines by quantitative reverse transcription–polymerase chain reaction and immunohistochemistry. In lung cancer cell lines that express netrin-1, the expression of netrin-1 was inhibited by using small interfering RNA (siRNA), or interference with netrin-1 was performed by treatment with a decoy recombinant DCC ectodomain protein (DCC-5Fbn). Cell death was monitored with a trypan blue exclusion assay or by measuring caspase-3 activity. The effect of netrin-1 interference on tumor growth was analyzed by DCC-5Fbn intratumoral or netrin-1 siRNA intraperitoneal injection in mice engrafted with lung cancer cell lines. All statistical tests were two-sided. High levels of netrin-1 were found in 43 of the 92 NSCLC tumor samples (47%). Interference with netrin-1 in human lung cancer cell lines was associated with UNC5H-mediated cell death in vitro (percentage of cell death in untreated and in DCC-5Fbn–treated cells = 8% and 26%, respectively, difference = 18%, 95% confidence interval [CI] = 10% to 26%; P = .049) and with lung tumor growth inhibition and/or regression in xenografted nude mice (12 mice in DCC-5Fbn–treated group and 13 mice in control group). Mean volume of control and DCC-5Fbn–treated tumors on day 46 was 489 and 84 mm ³ , respectively (difference = 404 mm ³ , 95% CI = 145 to 664 mm ³ ; P < .001). Almost half of the NSCLC tissue samples examined expressed high levels of netrin-1. Extracellular targeting of the interaction between netrin-1 and UNC5H may be a promising therapeutic approach for NSCLCs that express netrin-1.

The p33ING1 protein is a regulator of cell cycle, senescence, and apoptosis. Three alternatively spliced transcripts of p33ING1 encode p47ING1a, p33ING1b, and p24ING1c. We cloned an additional ING family member, p33ING2 / ING1L . Unlike p33ING1b, p33ING2 is induced by the DNA-damaging agents etoposide and neocarzinostatin. p33ING1b and p33ING2 negatively regulate cell growth and survival in a p53-dependent manner through induction of G ₁ -phase cell-cycle arrest and apoptosis. p33ING2 strongly enhances the transcriptional-transactivation activity of p53. Furthermore, p33ING2 expression increases the acetylation of p53 at Lys-382. Taken together, p33ING2 is a DNA damage-inducible gene that negatively regulates cell proliferation through activation of p53 by enhancing its acetylation.

A candidate tumor suppressor gene, p33ING1, was previously identified by using the genetic suppressor element methodology. p33ING1 cooperates with p53 and plays a significant role in p53-mediated cellular processes. Recently, we have identified p33ING2, which shows a sequence homology similar to p33ING1 and modulates p53 function. In the present study, we identified and characterized another ‘ING family’ gene. The estimated molecular weight of the encoded protein is 46.8 kDa, thus, we named it p47ING3. The p47ING3 gene is located at chromosome 7q31.3 and consists of 12 exons that encode 418 amino acids. A computational domain search revealed a C-terminal PHD-finger motif. Such motifs are common in proteins involved in chromatin remodeling. p47ING3 is highly expressed in some normal human tissues or organs, including the spleen, testis, skelet al muscle, and heart. p47ING3 expression levels varied among cancer cell lines. p47ING3 overexpression resulted in a decreased population of cells in S phase, a diminished colony-forming efficiency, and induced apoptosis in RKO cells, but not in RKO-E6 cells with inactivated p53. p47ING3 activates p53-transactivated promoters, including promoters of p21/waf1 and bax. Thus, we have isolated a novel ING family gene, p47ING3, which modulates p53-mediated transcription, cell cycle control, and apoptosis.

Bloom's syndrome is a rare autosomal recessive genetic disorder characterized by chromosomal aberrations, genetic instability, and cancer predisposition, all of which may be the result of abnormal signal transduction during DNA damage recognition. Here, we show that BLM is an intermediate responder to stalled DNA replication forks. BLM colocalized and physically interacted with the DNA damage response proteins 53BP1 and H2AX. Although BLM facilitated physical interaction between p53 and 53BP1, 53BP1 was required for efficient accumulation of both BLM and p53 at the sites of stalled replication. The accumulation of BLM/53BP1 foci and the physical interaction between them was independent of γ-H2AX. The active Chk1 kinase was essential for both the accurate focal colocalization of 53BP1 with BLM and the consequent stabilization of BLM. Once the ATR/Chk1- and 53BP1-mediated signal from replicational stress is received, BLM functions in multiple downstream repair processes, thereby fulfilling its role as a caretaker tumor suppressor.