Roles of Myc and Mad in cell cycle and apoptosis

The Myc network proteins are key mediators in regulation of cell growth, differentiation and apoptosis. They are basic region hehx-loop-helix/leucine zipper (bHLH/Zip) transcription factors that require hetero-dimerization with Max for specific DNA binding Mad family members are expressed primarily in differentiated tissues where they recruit histone deacetylase complexes via the mSin3 interaction domain (SID) to repress transcription of target genes and prevent cell growth In contrast, members of the Myc family activate target gene transcription by recruitment of histone acetyltransferases to their transcriptional activation domain (TAD), inducing proliferation and S phase entry. Myc activation also sensitizes cells to apoptosis in response to stress such as serum deprivation or exposure to cytotoxic drugs. However, mutations acquired during cancer therapy often block Mycdriven apoptosis, explaining the presence of activated myc in many human tumors. We have characterized the effects of Mad1 and Myc in cell growth control and in apoptosis induced by low serum, by anticancer drugs, or by differentiating agents. Using tet-mad1 inducible cells, we emphasized the important role for Mad1 in inhibition of cell proliferation in low serum, and showed a correlation with a reduced CDK2 activity In addition, Mad1 induction blocked cell cycle re-entry and resulted in reduced apoptosis in response to serum starvation and to the cytotoxic drug cisplatin. We demonstrated that these effects required transcriptional repression and suggest that Mad1 ensures cell survival and specialization by stabilizing quiescence and protecting against apoptosis during differentiation. To explore the effect of Myc on the cellular response to conventional chemotherapy, routinely used to complement surgery and radiation therapy when treating cancer patients, we used tet-myc inducible cells, together with Rat1 fibroblasts with different Myc status. In these model systems, we demonstrated that c-Myc enhanced the apoptosis induced by etoposide, doxorubicin, and cisplatin. Furthermore, we found that etoposide and doxorubicin signaling involved activation of pro-apoptotic Bax and of caspase 3 and 9. In addition, etoposide required proapoptotic PKCdelta for efficient apoptosis induction. We observed a similar Myc-dependence for efficient apoptosis induction by the chemotherapeutic agents camptothecin and paclitaxel. Apoptosis was enhanced both by c-Myc in Rat1 cells and by MYCN in neuroblastoma cells with conditional MYCN expression. While camptothecin signaling involved activation of Bax and caspases together with PKCdelta, our data suggest that paclitaxel induces apoptosis through a pathway distinct from mitochondria and PKCdelta signaling. Neither of the drugs affected Myc/Max DNAbinding, but camptothecin treatment reduced transactivation by several transcription factors, suggesting this as a mechanism for its effects. Taken together, our data establish the involvement of Bay, caspases, and PKCdelta signaling in Mycdependent apoptosis induced by etoposide and camptothecin, but not by cisplatin and paclitaxel. We also analyzed cellular differentiation and apoptosis in response to treatment with all-trans retinoic add (ATRA) and arsenic tdoxide (As2O3), used in treatment of acute myelocyfic leukemia- Although the fusion protein PML-RARalpha is a well established target for these drugs, additional mechanisms for their induction of differentiation and/or apoptosis are poorly characterized. For this purpose, we used the PML-RARalpha-negative promyelocytic leukemia cell fine HL60, and confirmed the connection between Myc expression and cellular differentiation status. We found that ATRA-induced terminal differentiation and apoptosis coincided with down-regulation of Myc, while the partially differentiated AS203 treated cells had a repressed, but not abolished, Myc expression. Myc was also present at the promoters of its target genes human telomerase reverse transcriptase (hTERT) and carbamoyltransferase-dihydroorotase (CAD) after exposure to AS203 but not ATRA, suggesting Myc as an important mediator in preventing terminal differentiation after AS203 treatment, possibly through activation of hTERT and CAD. In conclusion, characterization of the pathways for Myc-mediated apoptosis is essential in the venture to enable their reactivation in tumors overexpressing Myc and thus overcoming acquired drug-resistance. Therefore, Myc levels in human tumors should be considered for tailored treatment using anticancer drugs

Identification of cytotoxic drugs that selectively target tumor cells with MYC overexpression.

Expression of MYC is deregulated in a wide range of human cancers, and is often associated with aggressive disease and poorly differentiated tumor cells. Identification of compounds with selectivity for cells overexpressing MYC would hence be beneficial for the treatment of these tumors. For this purpose we used cell lines with conditional MYCN or c-MYC expression, to screen a library of 80 conventional cytotoxic compounds for their ability to reduce tumor cell viability and/or growth in a MYC dependent way. We found that 25% of the studied compounds induced apoptosis and/or inhibited proliferation in a MYC-specific manner. The activities of the majority of these were enhanced both by c-MYC or MYCN over-expression. Interestingly, these compounds were acting on distinct cellular targets, including microtubules (paclitaxel, podophyllotoxin, vinblastine) and topoisomerases (10-hydroxycamptothecin, camptothecin, daunorubicin, doxorubicin, etoposide) as well as DNA, RNA and protein synthesis and turnover (anisomycin, aphidicholin, gliotoxin, MG132, methotrexate, mitomycin C). Our data indicate that MYC overexpression sensitizes cells to disruption of specific pathways and that in most cases c-MYC and MYCN overexpression have similar effects on the responses to cytotoxic compounds. Treatment of the cells with topoisomerase I inhibitors led to down-regulation of MYC protein levels, while doxorubicin and the small molecule MYRA-A was found to disrupt MYC-Max interaction. We conclude that the MYC pathway is only targeted by a subset of conventional cytotoxic drugs currently used in the clinic. Elucidating the mechanisms underlying their specificity towards MYC may be of importance for optimizing treatment of tumors with MYC deregulation. Our data also underscores that MYC is an attractive target for novel therapies and that cellular screenings of chemical libraries can be a powerful tool for identifying compounds with a desired biological activity

Myc Is Required for Activation of the ATM-Dependent Checkpoints in Response to DNA Damage

Background: The MYC protein controls cellular functions such as differentiation, proliferation, and apoptosis. In response to genotoxic agents, cells overexpressing MYC undergo apoptosis. However, the MYC-regulated effectors acting upstream of the mitochondrial apoptotic pathway are still unknown. Principal Findings: In this study, we demonstrate that expression of Myc is required to activate the Ataxia telangiectasia mutated (ATM)-dependent DNA damage checkpoint responses in rat cell lines exposed to ionizing radiation (IR) or the bacterial cytolethal distending toxin (CDT). Phosphorylation of the ATM kinase and its downstream effectors, such as histone H2AX, were impaired in the myc null cell line HO15.19, compared to the myc positive TGR-1 and HOmyc3 cells. Nuclear foci formation of the Nijmegen Breakage Syndrome (Nbs) 1 protein, essential for efficient ATM activation, was also reduced in absence of myc. Knock down of the endogenous levels of MYC by siRNA in the human cell line HCT116 resulted in decreased ATM and CHK2 phosphorylation in response to irradiation. Conversely, cell death induced by UV irradiation, known to activate the ATR-dependent checkpoint, was similar in all the cell lines, independently of the myc status. Conclusion: These data demonstrate that MYC contributes to the activation of the ATM-dependent checkpoint responses, leading to cell death in response to specific genotoxic stimuli.Swedish Cancer SocietySwedish Research Counci

<i>myc</i> deletion prevents p53 stabilization in response to DNA damage.

<p><b>A</b>) TGR-1 and the <i>myc</i> reconstituted HOmyc3 cells were left untreated or exposed to IR (20 Gy) for the indicated periods of time. The levels of p53 were assessed by western blot analysis. <b>B</b>) TGR-1, the <i>myc</i> reconstituted HOmyc3 and the HO15.19 cells were left untreated or exposed to IR (20 Gy) for the indicated periods of time. The levels of p53 were assessed by western blot analysis. Actin was used as an internal loading control. One out of three independent experiments is shown.</p

<i>myc</i> deletion delays cell death upon irradiation.

<p><b>A</b>) The levels of the endogenous Myc protein were assessed by immunoprecipitation followed by western blot analysis in TGR-1, the <i>myc</i> reconstituted cells HOmyc3, and the <i>myc</i> null HO15.19 cells using α-Myc antibodies. Expression of actin in total cell lysates was used as control. <b>B</b>) TGR-1, the <i>myc</i> reconstituted cells HOmyc3, and the <i>myc</i> null HO15.19 cells were left untreated or irradiated (20Gy) and further incubated in complete medium for the indicated periods of time. Analysis of the cell cycle distribution was assessed by PI staining and flow cytometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008924#s2" target="_blank">Material and Methods</a>. One out of four independent experiments is shown.</p

Activation of ATM and ATM-dependent responses upon induction of DNA damage is Myc dependent.

<p>TGR-1, HOmyc3, and HO15.19 cells were left untreated or exposed to IR (20Gy), and further incubated in complete medium for 2h. The levels of phospho-ATM and phospho-H2AX (γH2AX) were assessed by western blot analysis. Actin and total ATM were used as internal loading controls. One of three experiments is shown.</p

<i>myc</i> deletion does not alter cell death kinetics upon UV irradiation.

<p>TGR-1, the <i>myc</i> reconstituted cells HOmyc3, and the <i>myc</i> null HO15.19 cells were left untreated or exposed to UV irradiation and further incubated in complete medium for the indicated periods of time. <b>A</b>) Analysis of the cell cycle distribution assessed by PI staining and flow cytometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008924#s2" target="_blank">Material and Methods</a>. <b>B</b>) Phase contrast micrographs of the cells taken at the indicated time points (Magnification 40×). One out of three independent experiments is shown.</p