The wild-type p53-induced phosphatase PPM1D (or Wip1) is a serine/threonine phosphatase that is transcriptionally upregulated by p53 following ultraviolet and ionizing radiation. PPM1D is an oncogene in transformation assays and is amplified or overexpressed in several human tumor types. Here, we demonstrate that PPM1D interacts with the nuclear isoform of uracil DNA glycosylase, UNG2, and suppresses base excision repair (BER). Point mutations that inactivate PPM1D phosphatase activity abrogate BER suppression, indicating that dephosphorylation by PPM1D is important for BER inhibition. We have identified UNG2 phosphorylation sites at threonines 6 and 126 that exhibit enhanced phosphorylation following UV irradiation. The UV-induced phosphorylated forms of UNG2 are more active than nonphosphorylated forms in mediating uracil-associated DNA cleavage. PPM1D dephosphorylation of UNG2 at phosphothreonine 6 is associated with reduced UNG2 activity. Thus, PPM1D may inhibit BER by dephosphorylating UNG2 to facilitate its inactivation after completion of DNA repair.

The melanoma differentiation-associated gene-7 (mda-7/IL24) is a unique member of the IL-10 family of cytokines, with ubiquitous tumor cell proapoptotic activity. Transduction of tumor or normal cells with the mda-7 gene results in secretion of glycosylated MDA-7 protein. Recent data indicate that secreted MDA-7 protein functions as a pro-Th1 cytokine and as a potent antiangiogenic molecule. MDA-7 protein binds two distinct type II cytokine heterodimeric receptor complexes, IL-20R1/IL-20R2 (type 1 IL-20R) and IL-22R1/IL-20R2 (type 2 IL-20R). In this study we analyzed the activity of glycosylated secreted MDA-7 against human melanoma cells. MDA-7 protein induces phosphorylation and nuclear translocation of STAT3 in melanoma cells via both type 1 and type 2 IL-20R. MDA-7 induces dose-dependent cell death in melanoma tumor cells. MDA-7 receptor engagement results in up-regulation of BAX and subsequent apoptosis induction; this effect is mediated by STAT3-independent signaling. Additional IL-10 family members (IL-10, -19, -20, and -22) also activate STAT3; however, these ligands do not activate death pathways in melanoma. In normal cells, MDA-7 can bind to its cognate receptors and induce phosphorylation of STAT3, without cytotoxic sequelae. This study defines a tumor-selective cytotoxic bystander role for secreted MDA-7 protein and identifies a novel receptor-mediated, STAT3-independent, and PKR-independent death pathway.

The melanoma differentiation-associated gene (mda-7; approved gene symbol IL24) is a tumor suppressor gene whose protein expression in normal cells is restricted to the immune system and to melanocytes. Recent studies have shown that mda-7 gene transfer inhibits cell growth and induces apoptosis in melanoma, lung cancer, breast cancer, and other tumor types through activation of various intracellular signaling pathways. In the current study, we demonstrate that Ad-mda7 transduction of human pancreatic cancer cells results in G2/M cell cycle arrest and cell killing. Cytotoxicity is mediated via apoptosis in a time- and dose-dependent manner. Tumor cell killing correlates with regulation of proteins involved in the Wnt and PI3K pathways: beta-catenin, APC, GSK-3, JNK, and PTEN. Additionally, we identify bystander cell killing activated by exposure of pancreatic tumor cells to secreted human MDA-7 protein. In pancreatic tumor cells, exogenous MDA-7 protein activates STAT3 and kills cells via engagement of IL-20 receptors. The specificity of bystander killing is demonstrated using neutralizing anti-MDA-7 antibodies and anti-receptor antibodies, which inhibit the apoptotic effects. In sum, we show that Ad-mda7 is able to induce growth inhibition and apoptosis in pancreatic cancer cells via inhibition of the Wnt/PI3K pathways and identify a novel bystander mechanism of MDA-7 killing in pancreatic cancer that functions via IL-20 receptors.

The melanoma differentiation-associated gene-7 (mda-7/IL-24) is a unique member of the interleukin 10 (IL-10) family of cytokines, with ubiquitous tumor cell pro-apoptotic activity. Recent data have shown that IL-24 is secreted as a glycosylated protein and functions as a pro-Th1 cytokine and as a potent anti-angiogenic molecule. In this study, we analyzed the activity of Ad-mda7 and its protein product, secreted IL-24, against human breast cancer cells. We show that Ad-mda7 transduction of human breast cancer cells results in G(2)/M phase cell cycle arrest and apoptotic cell death, which correlates with secretion of IL-24 protein. Neutralizing antibody against IL-24 significantly inhibited Ad-mda7 cytotoxicity. IL-24 and IL-10 both engage their cognate receptors on breast cancer cells resulting in phosphorylation and activation of STAT3, however, IL-10 receptor binding failed to induce cell killing, indicating that tumor cell killing by IL-24 is independent of STAT3 phosphorylation. Treatment with exogenous IL-24 induced apoptosis in breast cancer cells and this effect was abolished by addition of anti-IL-24 antibody or anti-IL-20R1, indicating that bystander cell killing is mediated via IL-24 binding to the IL-20R1/IL-20R2 heterodimeric receptor complex. Co-administration of the related cytokine IL-10 inhibited killing mediated by IL-24 and concomitantly inhibited IL-24 mediated up-regulation of the tumor suppressor proteins, p53 and p27(Kip1). In summary, we have defined a tumor-selective cytotoxic bystander role for secreted IL-24 protein and identified a novel receptor-mediated death pathway in breast cancer cells, wherein the related cytokines IL-24 and IL-10 exhibit antagonistic activity.

No abstract

Melanoma is the most malignant of skin cancers, highly resistant to chemotherapy and radiotherapy. Temozolomide, a promising new derivative of dacarbazine, is currently being tested for treatment of metastatic melanoma. Resistance to alkylating agents such as temozolomide correlates with increased expression of DNA repair protein O 6 -methylguanine-DNA methyltransferase (MGMT). Interleukin-24 (IL-24; mda-7) is a tumor suppressor cytokine that selectively inhibits tumor cell growth by inducing apoptosis and cell cycle arrest in melanoma cell lines and solid tumors. This tumor-selective activity has been observed in multiple preclinical animal models and in clinical trials. In this study, we analyzed the ability of Ad-IL-24 and its protein product, IL-24, to overcome temozolomide resistance in human melanoma cells. We have shown that Ad-IL-24 via exogenous IL-24 protein induces combinatorial synergy of temozolomideinduced cell killing in temozolomide-resistant melanoma cells by inhibition of MGMT. Neutralizing antibodies against IL-24 or its receptors significantly blocked the apoptotic activity of IL-24 + MGMT treatment. We show that accumulation of functional p53 is essential for IL-24-induced down-regulation of MGMT. Using either MGMT small interfering RNA, p53 small interfering RNA, or a p53 dominant-negative mutant to block MGMT protein expression resulted in increased sensitization to temozolomide. However, MGMT blockade in combination with IL-24 + temozolomide resulted in loss of combinatorial synergy, indicating that MGMT expression is required for the reversal of temozolomide resistance in melanoma cells. This study shows that IL-24 can play a significant role in overcoming temozolomide resistance and that the clinical efficacy of temozolomide may be improved by using a biochemotherapy combination with IL-24. [Mol Cancer Ther 2008;7(12):3842 -51]

Current therapies used in the treatment of breast cancer are limited by systemic toxicity, rapid drug metabolism and intrinsic and acquired drug resistance. We have previously shown that adenoviral-mediated transfer of the melanoma differentiation-associated gene-7 (mda-7) elicits growth inhibition and apoptosis in various tumor types. Here, we evaluate the effects of Ad-mda7, alone and in combination with other therapies, against a panel of nine breast tumor cell lines and their normal counterparts; we report selective Ad-mda7-mediated p53-independent growth inhibition, G2/M cell cycle arrest, and apoptosis. In vivo, Ad-mda7 induced p53-independent tumor growth inhibition (Po0.004) in multiple xenograft models. We then evaluated the combination of Admda7 with agents commonly used to treat breast cancer: radiotherapy (XRT), Tamoxifen, Taxotere, Adriamycin, and Herceptin. These agents exhibit diverse modes of action, including formation of bulky adducts, inhibition of DNA replication (Adriamycin, XRT), damage to microtubules (Taxotere), nonsteroidal estrogen antagonists (Tamoxifen), or Her2/neu receptor blockade (Herceptin). Treated with conventional anticancer drugs or radiation, MDA-7-expressing cells display additive or synergistic cytotoxicity and apoptosis that correlates with decreased BCL-2 expression and BAX upregulation. In vivo, animals that received Ad-mda7 and XRT underwent significant reduction of tumor growth (Po0.002). This is the first report of the synergistic effects of Ad-mda7 combined with chemotherapy or radiotherapy on human breast carcinoma cells.

We compared the effects of glial cell line-derived neurotrophic factor (GDNF) on dorsal root ganglion (DRG) sensory neurons to that of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT-3). All of these factors were retrogradely transported to subpopulations of sensory neuron cell bodies in the L4/ L5 DRG of neonatal rats. The size distribution of 125I-GDNF-labeled neurons was variable and consisted of both small and large DRG neurons (mean of 506.60 microns2). 125I-NGF was preferentially taken up by small neurons with a mean cross-sectional area of 383.03 microns2. Iodinated BDNF and NT-3 were transported by medium to large neurons with mean sizes of 501.48 and 529.27 microns2, respectively. A neonatal, sciatic nerve axotomy-induced cell death model was used to determine whether any of these factors could influence DRG neuron survival in vivo. GDNF and NGF rescued nearly 100% of the sensory neurons. BDNF and NT-3 did not promote any detectable level of neuronal survival despite the fact that they underwent retrograde transport. We examined the in vitro survival-promoting ability of these factors on neonatal DRG neuronal cultures derived from neonatal rats. GDNF, NGF, and NT-3 were effective in vitro, while BDNF was not. The range of effects seen in the models described here underscores the importance of testing neuronal responsiveness in more than one model. The biological responsiveness of DRG neurons to GDNF in multiple models suggests that this factor may play a role in the development and maintenance of sensory neurons.