Replication protein A (RPA) is a DNA single‐strand binding protein essential for DNA replication, recombination and repair. In human cells treated with the topoisomerase inhibitors camptothecin or etoposide (VP‐16), we find that RPA2, the middle‐sized subunit of RPA, becomes rapidly phosphorylated. This response appears to be due to DNA‐dependent protein kinase (DNA‐PK) and to be independent of p53 or the ataxia telangiectasia mutated (ATM) protein. RPA2 phosphorylation in response to camptothecin required ongoing DNA replication. Camptothecin itself partially inhibited DNA synthesis, and this inhibition followed the same kinetics as DNA‐PK activation and RPA2 phosphorylation. DNA‐PK activation and RPA2 phosphorylation were prevented by the cell‐cycle checkpoint abrogator 7‐hydroxystaurosporine (UCN‐01), which markedly potentiates camptothecin cytotoxicity. The DNA‐PK catalytic subunit (DNA‐PKcs) was found to bind RPA which was replaced by the Ku autoantigen upon camptothecin treatment. DNA‐PKcs interacted directly with RPA1 in vitro. We propose that the encounter of a replication fork with a topoisomerase–DNA cleavage complex could lead to a juxtaposition of replication fork‐associated RPA and DNA double‐strand end‐associated DNA‐PK, leading to RPA2 phosphorylation which may signal the presence of DNA damage to an S‐phase checkpoint mechanism. Keywords: camptothecin/DNA damage/DNA‐dependent protein kinase/RPA2 phosphorylation

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Salivary gland tumors (SGT) are a group of highly heterogeneous head and neck malignancies with widely varied clinical outcomes and no standard effective treatments. The CRTC1-MAML2 fusion oncogene, encoded by a recurring chromosomal translocation t(11;19)(q14-21;p12-13), is a frequent genetic alteration found in >50% of mucoepidermoid carcinomas (MEC), the most common malignant SGT. In this study, we aimed to define the role of the CRTC1-MAML2 oncogene in the maintenance of MEC tumor growth and to investigate critical downstream target genes and pathways for therapeutic targeting of MEC. By performing gene expression analyses and functional studies via RNA interference and pharmacological modulation, we determined the importance of the CRTC1-MAML2 fusion gene and its downstream AREG-EGFR signaling in human MEC cancer cell growth and survival in vitro and in vivo using human MEC xenograft models. We found that CRTC1-MAML2 fusion oncogene was required for the growth and survival of fusion-positive human MEC cancer cells in vitro and in vivo. The CRTC1-MAML2 oncoprotein induced the upregulation of the epidermal growth factor receptor (EGFR) ligand Amphiregulin (AREG) by co-activating the transcription factor CREB, and AREG subsequently activated EGFR signaling in an autocrine manner that promoted MEC cell growth and survival. Importantly, CRTC1-MAML2-positive MEC cells were highly sensitive to EGFR signaling inhibition. Therefore, our study revealed that aberrantly activated AREG-EGFR signaling is required for CRTC1-MAML2-positive MEC cell growth and survival, suggesting that EGFR-targeted therapies will benefit patients with advanced, unresectable CRTC1-MAML2-positive MEC.

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Protein phosphorylation plays an important role in signal transduction, but its involvement in apoptosis still remains unclear. In this report, the p53-null human leukemia HL60 cells were used to investigate phosphorylation and degradation of lamin B during apoptosis. We found that lamin B was phosphorylated within 1 h after addition of the DNA topoisomerase I inhibitor, camptothecin, and that lamin B phosphorylation preceded lamin B degradation and DNA fragmentation. Using a cell-free system we also found that cytosol from camptothecin-treated cells induced lamin B phosphorylation and degradation in isolated nuclei from untreated HL60 cells. Lamin B phosphorylation was prevented by the protein kinase C (PKC) inhibitor 7-hydroxystaurosporine (UCN-01) but not by the Cdc2 inhibitor, flavopiridol. Phosphorylation of lamin B was inhibited by immunodepletion of PKC␣ from activated cytosol and was restored by addition of purified PKC␣. PKC␣ activity also increased rapidly as lamin B was phosphorylated after initiation of the apoptotic response in HL60 cells. These data suggest that lamin B is phosphorylated by PKC␣ and proteolyzed before DNA fragmentation in HL60 cells undergoing apoptosis.The nuclear lamins are karyophilic proteins located at the nucleoplasmic surface of the inner nuclear membrane where they assemble in a polymeric structure referred to as the nuclear lamina (for review, see Refs. 1-3). Lamins belong to the family of intermediate filaments, which share a tripartite organization consisting of a central ␣-helical rod domain of conserved size, flanked by N-and C-terminal non-␣-helical end domains of variable size and sequence (see Fig. 9). The lamina has been suggested to serve as a major chromatin anchoring site of nuclear scaffold-associated regions during interphase and possibly to be involved in organizing higher order chromatin domains. The lamina is a dynamic structure regulated by phosphorylation. Phosphorylation by p34 cdc2 kinase is key to the dissolution of the nuclear lamina during mitosis. Other lamin kinases include mitogen-associated protein kinases, c-AMP-dependent protein kinase (PKA) 1 and protein kinase C (PKC) (1-3). Major PKC phosphorylation sites have been mapped to serine residues located in close proximity to the nuclear localization signal in the C-terminal non-␣-helical region, and phosphorylation of these residues interferes with the nuclear transport of lamin B (2). The p34 cdc2 phosphorylation sites are on both sides of the central ␣-helical rod domain. While many mammalian cells contains three distinct lamins (lamins A, B, and C), human leukemia HL60 cells express primarily lamin B (4).Lamin proteolysis during apoptosis has been reported in various cell lines treated with different stimuli. In human leukemia HL60 cells treated with etoposide (VP-16) (5) or camptothecin (CPT) (6), apoptosis is accompanied by diminished levels of lamin B. Etoposide is a topoisomerase II inhibitor (7) and CPT a topoisomerase I inhibitor (8). Both drugs are effective anti-cancer agents. Lamin B1...

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We previously demonstrated that the anticancer agent and protein kinase C (PKC) inhibitor 7-hydroxystaurosporine (UCN-01) induces apoptosis independently of p53 and protein synthesis in HL60 cells. We now report the associated changes of PKC isoforms. PKC␣, ␤I, ␤II, ␦, and activities were measured after immunoprecipitation of cytosols from UCN-01-treated HL60 cells. UCN-01 had no effect on PKC and inhibited kinase activity of PKC␤I, ␤II, and ␦. PKC␣ activity was initially inhibited at 1 h, and subsequently increased as cells underwent apoptosis 3 h after the beginning of UCN-01 treatment. Camptothecin (CPT) and etoposide (VP-16) also markedly enhanced PKC␣ activity during apoptosis in HL60 cells. However, CPT did not affect PKC␤I, ␤II and , and activated PKC␦. PKC␣ activation was not due to increased protein levels or proteolytic cleavage but was associated with PKC␣ autophosphorylation in vitro and increased phosphorylation in vivo. We also found that not only PKC ␦ but also PKC ␤I was proteolytically activated in HL60 cells during apoptosis. The PKC␣ activation and hyperphosphorylation were abrogated by N-benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethylketone (z-VAD-fmk) under conditions that abrogated apoptosis. z-VAD-fmk also prevented PKC␦ and ␤I proteolytic activation. Together these findings suggest that caspases regulate PKC activity during apoptosis in HL60 cells. At least two modes of activation were observed: hyperphosphorylation for PKC␣ and proteolytic activation for PKC ␦ and ␤I.

MYB activation is proposed to underlie development of adenoid cystic cancer (ACC), an aggressive salivary gland tumor with no effective systemic treatments. To discover druggable targets for ACC, we performed global mRNA/miRNA analyses of 12 ACC with matched normal tissues, and compared these data with 14 mucoepidermoid carcinomas (MEC) and 11 salivary adenocarcinomas (ADC). We detected a unique ACC gene signature of 1160 mRNAs and 22 miRNAs. MYB was the top-scoring gene (18-fold induction), however we observed the same signature in ACC without detectable MYB gene rearrangements. We also found 4 ACC tumors (1 among our 12 cases and 3 from public databases) with negligible MYB expression that retained the same ACC mRNA signature including over-expression of extracellular matrix (ECM) genes. Integration of this signature with somatic mutational analyses suggests that NOTCH1 and RUNX1 participate with MYB to activate ECM elements including the VCAN/HAPLN1 complex. We observed that forced MYB-NFIB expression in human salivary gland cells alters cell morphology and cell adhesion in vitro and depletion of VCAN blocked tumor cell growth of a short-term ACC tumor culture. In summary, we identified a unique ACC signature with parallel MYB-dependent and independent biomarkers and identified VCAN/HAPLN1 complexes as a potential target.