Roles of Myc and Mad in cell cycle and apoptosis

Abstract

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.