The epidermal growth factor receptor (EGFR) is frequently dysregulated in malignant glioma that leads to increased resistance to cancer therapy. Upregulation of wild type or expression of mutant EGFR is associated with tumor radioresistance and poor clinical outcome. EGFR variant III (EGFRvIII) is the most common EGFR mutation in malignant glioma. Radioresistance is thought to be, at least in part, the result of a strong cytoprotective response fueled by signaling via AKT and ERK that is heightened by radiation in the clinical dose range. Several groups including ours have shown that this response may modulate DNA repair. Herein, we show that expression of EGFRvIII promoted γ-H2AX foci resolution, a surrogate for double-strand break (DSB) repair, and thus enhanced DNA repair. Conversely, small molecule inhibitors targeting EGFR, MEK, and the expression of dominant-negative EGFR (EGFR-CD533) significantly reduced the resolution of γ-H2AX foci. When homologous recombination repair (HRR) and non-homologous end joining (NHEJ) were specifically examined, we found that EGFRvIII stimulated and CD533 compromised HRR and NHEJ, respectively. Furthermore, NHEJ was blocked by inhibitors of AKT and ERK signaling pathways. Moreover, expression of EGFRvIII and CD533 increased and reduced, respectively, the formation of phospho-DNA-PKcs and -ATM repair foci, and RAD51 foci and expression levels, indicating that DSB repair is regulated at multiple levels. Altogether, signaling from EGFR and EGFRvIII promotes both HRR and NHEJ that is likely a contributing factor towards the radioresistance of malignant gliomas.

SummaryMale gametogenesis occurs directly after uptake of malaria parasites by the mosquito vector and leads to the release of eight nucleated flagellar gametes. Here, we report that one of the two parasite actin isoforms, named actin II, is essential for this process. Disruption of actin II in Plasmodium berghei resulted in viable asexual blood stages, but male gametogenesis was specifically inhibited. Upon activation, male gametocyte DNA was replicated normally and axonemes assembled, but egress from the host cell was inhibited, and axoneme motility abolished. The major actin isoform, actin I, displayed dual localization to the cytoplasm and the nucleus in male gametocytes. After activation actin I was found to be restricted to the cytoplasm. In actII(-) mutant parasites, this re-localization was abolished and actin I remained in both cellular compartments. These findings reveal vital and pleiotropic functions for the actin II isoform in male gametogenesis of the malaria parasite.

SummarySuccessful gametogenesis of the malaria parasite depends on egress of the gametocytes from the erythrocytes within which they developed. Egress entails rupture of both the parasitophorous vacuole membrane and the erythrocyte plasma membrane, and precedes the formation of the motile flagellated male gametes in a process called exflagellation. We show here that egress of the male gametocyte depends on the function of a perforin-like protein, PPLP2. A mutant of Plasmodium berghei lacking PPLP2 displayed abnormal exflagellation; instead of each male gametocyte forming eight flagellated gametes, it produced gametocytes with only one, shared thicker flagellum. Using immunofluorescence and transmission electron microscopy analysis, and phenotype rescue with saponin or a pore-forming toxin, we conclude that rupture of the erythrocyte membrane is blocked in the mutant. The parasitophorous vacuole membrane, on the other hand, is ruptured normally. Some mutant parasites are still able to develop in the mosquito, possibly because the vigorous motility of the flagellated gametes eventually leads to escape from the persisting erythrocyte membrane. This is the first example of a perforinlike protein in Plasmodium parasites having a role in egress from the host cell and the first parasite protein shown to be specifically required for erythrocyte membrane disruption during egress.

Ionizing radiation (IR) triggers many signaling pathways primarily originating from either damaged DNA or non-nuclear sources such as growth factor receptors. Thus, to study the DNA damage-induced signaling component alone by irradiation would be a challenge. To generate DNA double-strand breaks (DSBs) and minimize non-nuclear signaling, human cancer cells having bromodeoxyuridine (BrdU) - substituted DNA were treated with the photosensitizer Hoechst 33258 followed by long wavelength UV (UV-A) treatment (BrdU photolysis). BrdU photolysis resulted in well-controlled, dose- dependent generation of DSBs equivalent to radiation doses between 0.2 - 20 Gy, as determined by pulsed-field gel electrophoresis, and accompanied by dose-dependent ATM (ser-1981), H2AX (ser-139), Chk2 (thr-68), and p53 (ser-15) phosphorylation. Interestingly, low levels (≤ 2 Gy equivalents) of BrdU photolysis stimulated ERK phosphorylation whereas higher (> 2 Gy eq.) resulted in ERK dephosphorylation. ERK phosphorylation was ATM-dependent whereas dephosphorylation was ATM-independent. The ATM-dependent increase in ERK phosphorylation was also seen when DSBs were generated by transfection of cells with an EcoRI expression plasmid or by electroporation of EcoRI enzyme. Furthermore, AKT was critical for transmitting the DSB signal to ERK. Altogether, our results show that low levels of DSBs trigger ATM- and AKT-dependent ERK pro-survival signaling and increased cell proliferation whereas higher levels result in ERK dephosphorylation consistent with a dose-dependent switch from pro-survival to anti-survival signaling.