COVID-RO study: the radiation oncology practice at times of COVID-19 outbreak — international survey

BackgroundRadiation therapy (RT), an essential treatment of cancer, involves multiple hospital visits. We hypothesized that radiation departments would adjust their work patterns and RT protocols in response to the SARS-CoV-2 pandemic.Materials and methodsAn electronic survey was sent during April 2020 to an international sample of radiation oncologists. The survey explored various aspects of departmental preparedness, and changes to their institutional RT protocols.ResultsA total of 68 radiation oncologists from 13 countries answered the survey. Healthcare systems were at least moderately affected in 76%. Most institutes appeared well prepared for the outbreak: regarding the availability of personal protective equipment, tests, and telemedicine/videoconference facilities. Screening for SARS-CoV-2 was applied in 59% of responders. Modification of RT protocols were minor in 66%, significant in 19% and no changes made in 15%. The extent to which protocols were modified correlated with overall healthcare disruption (p = 0.028). Normal fractionation was recommended to continue in 83% and 85% of head & neck, and cervical cancers vs. 64% of lung cancers (p = 0.001).In case the pandemic worsens, there was strong agreement to prioritize RT for aggressive cancers (80%), delay RT for slow-growing tumors (78%) and change to evidance-based hypofractionations protocols (79.4%). The option of delayed/omitted adjuvant RT (not site specific) was selected in 47%.ConclusionThis international survey concludes that, by making significant organizational adjustments and minor protocol modifications, RT may be safely continued during this pandemic. If the crisis worsens, there was strong agreement to continue the treatment of aggressive tumors and utilize evidence-based hypofractionated protocols.     

YopJ-induced caspase-1 activation in Yersinia-infected macrophages: independent of apoptosis, linked to necrosis, dispensable for innate host defense

Yersinia outer protein J (YopJ) is a type III secretion system (T3SS) effector of pathogenic Yersinia (Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis) that is secreted into host cells. YopJ inhibits survival response pathways in macrophages, causing cell death. Allelic variation of YopJ is responsible for differential cytotoxicity in Yersinia strains. YopJ isoforms in Y. enterocolitica O:8 (YopP) and Y. pestis KIM (YopJ(KIM)) strains have high cytotoxic activity. In addition, YopJ(KIM)-induced macrophage death is associated with caspase-1 activation and interleukin-1β (IL-1β secretion. Here, the mechanism of YopJ(KIM)-induced cell death, caspase-1 activation, and IL-1β secretion in primary murine macrophages was examined. Caspase-3/7 activity was low and the caspase-3 substrate poly (ADP-ribose) polymerase (PARP) was not cleaved in Y. pestis KIM5-infected macrophages. In addition, cytotoxicity and IL-1β secretion were not reduced in the presence of a caspase-8 inhibitor, or in B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax)/Bcl-2 homologous antagonist/killer (Bak) knockout macrophages, showing that YopJ(KIM)-mediated cell death and caspase-1 activation occur independent of mitochondrial-directed apoptosis. KIM5-infected macrophages released high mobility group protein B1 (HMGB1), a marker of necrosis, and microscopic analysis revealed that necrotic cells contained active caspase-1, indicating that caspase-1 activation is associated with necrosis. Inhibitor studies showed that receptor interacting protein 1 (RIP1) kinase and reactive oxygen species (ROS) were not required for cytotoxicity or IL-β release in KIM5-infected macrophages. IL-1β secretion was reduced in the presence of cathepsin B inhibitors, suggesting that activation of caspase-1 requires cathepsin B activity. Ectopically-expressed YopP caused higher cytotoxicity and secretion of IL-1β in Y. pseudotuberculosis-infected macrophages than YopJ(KIM). Wild-type and congenic caspase 1 knockout C57BL/6 mice were equally susceptible to lethal infection with Y. pseudotuberculosis ectopically expressing YopP. These data suggest that YopJ-induced caspase-1 activation in Yersinia-infected macrophages is a downstream consequence of necrotic cell death and is dispensable for innate host resistance to a strain with enhanced cytotoxicity.     

X-linked Inhibitor of Apoptosis Protein promotes the degradation of its antagonist, the pro-apoptotic ARTS protein

ARTS (Sept4_i2) is a mitochondrial pro-apoptotic tumor suppressor protein. In response to apoptotic signals, ARTS translocates to the cytosol where it promotes caspase activation through caspase de-repression and proteasome mediated degradation of X-linked Inhibitor of Apoptosis Protein (XIAP). Here we show that XIAP regulates the levels of ARTS by serving as its ubiquitin ligase, thereby providing a potential feedback mechanism to protect against unwanted apoptosis. Using both in vitro and in vivo ubiquitination assays we found that ARTS is directly ubiquitinated by XIAP. Moreover, we found that XIAP-induced ubiquitination and degradation is prevented by removal of the first four amino acids in the N-terminus of ARTS, which contains a single lysine residue at position 3. Thus, this lysine at position 3 is a likely target for ubiquitination by XIAP. Importantly, although the stabilized ARTS lacking its first 4 residues binds XIAP as well as the full length ARTS, it is more potent in promoting apoptosis than the full length ARTS. This suggests that increased stability of ARTS has a significant effect on its ability to induce apoptosis. Collectively, our data reveal a mutual regulatory mechanism by which ARTS and XIAP control each other's levels through the ubiquitin proteasome system.     

Phosphorylation of protein kinase Cdelta on distinct tyrosine residues induces sustained activation of Erk1/2 via down-regulation of MKP-1: role in the apoptotic effect of etoposide

The mechanism underlying the important role of protein kinase Cdelta (PKCdelta) in the apoptotic effect of etoposide in glioma cells is incompletely understood. Here, we examined the role of PKCdelta in the activation of Erk1/2 by etoposide. We found that etoposide induced persistent activation of Erk1/2 and nuclear translocation of phospho-Erk1/2. MEK1 inhibitors decreased the apoptotic effect of etoposide, whereas inhibitors of p38 and JNK did not. The activation of Erk1/2 by etoposide was downstream of PKCdelta since the phosphorylation of Erk1/2 was inhibited by a PKCdelta-KD mutant and PKCdelta small interfering RNA. We recently reported that phosphorylation of PKCdelta on tyrosines 64 and 187 was essential for the apoptotic effect of etoposide. Using PKCdeltatyrosine mutants, we found that the phosphorylation of PKCdeltaon these tyrosine residues, but not on tyrosine 155, was also essential for the activation of Erk1/2 by etoposide. In contrast, nuclear translocation of PKCdelta was independent of its tyrosine phosphorylation and not necessary for the phosphorylation of Erk1/2. Etoposide induced down-regulation of kinase phosphatase-1 (MKP-1), which correlated with persistent phosphorylation of Erk1/2 and was dependent on the tyrosine phosphorylation of PKCdelta. Moreover, silencing of MKP-1 increased the phosphorylation of Erk1/2 and the apoptotic effect of etoposide. Etoposide induced polyubiquitylation and degradation of MKP-1 that was dependent on PKCdelta and on its tyrosine phosphorylation. These results indicate that distinct phosphorylation of PKCdeltaon tyrosines 64 and 187 specifically activates the Erk1/2 pathway by the down-regulation of MKP-1, resulting in the persistent phosphorylation of Erk1/2 and cell apoptosis.     

A biochemical logic gate using an enzyme and its inhibitor. Part II: The logic gate

Enzyme-Based Logic Gates (ENLOGs) are key components in bio-molecular systems for information processing. This report and the previous one in this series address the characterization of two bio-molecular switching elements, namely the alpha-chymotrypsin (alphaCT) derivative p-phenylazobenzoyl-alpha-chymotrypsin (PABalphaCT) and its inhibitor (proflavine), as well as their assembly into a logic gate.The experimental output of the proposed system is expressed in terms of enzymic activity and this was translated into logic output (i.e. "1" or "0") relative to a predetermined threshold value. We have found that an univalent link exists between the dominant isomers of PABalphaCT (cis or trans), the dominant form of either acridine (proflavine) or acridan and the logic output of the system. Thus, of all possible combinations, only the trans-PABalphaCT and the acridan lead to an enzymic activity that can be defined as logic output "1". The system operates under the rules of Boolean algebra and performs as an "AND" logic gate.     

Synapsis-defective mutants reveal a correlation between chromosome conformation and the mode of double-strand break repair during Caenorhabditis elegans meiosis

SYP-3 is a new structural component of the synaptonemal complex (SC) required for the regulation of chromosome synapsis. Both chromosome morphogenesis and nuclear organization are altered throughout the germlines of syp-3 mutants. Here, our analysis of syp-3 mutants provides insights into the relationship between chromosome conformation and the repair of meiotic double-strand breaks (DSBs). Although crossover recombination is severely reduced in syp-3 mutants, the production of viable offspring accompanied by the disappearance of RAD-51 foci suggests that DSBs are being repaired in these synapsis-defective mutants. Our studies indicate that once interhomolog recombination is impaired, both intersister recombination and nonhomologous end-joining pathways may contribute to repair during germline meiosis. Moreover, our studies suggest that the conformation of chromosomes may influence the mode of DSB repair employed during meiosis.     

Differential expression of genes coding for EGF-like factors and ADAMTS1 following gonadotropin stimulation in normal and transformed human granulosa cells

We have demonstrated previously that the synthesis of epiregulin and amphiregulin, of the EGF-like growth factor family, is stimulated by luteinizing hormone in human follicular (granulosa) cells obtained from in vitro fertilization program. In the present work, we demonstrate that H89, a PKA inhibitor, attenuated the expression of these growth factors both in the mRNA and the protein levels, suggesting PKA involvement in this signaling pathway. SV40-transformed human granulosa cells showed higher basal levels of epiregulin and amphiregulin than normal cells, which were still elevated following cAMP stimulation by Forskolin. Cleavage by a disintegrin and metalloproteinases (ADAMs) is essential for activation of these growth factors, allowing their interaction with EGF receptor. Expression of ADAMTS1 and ADAM12 was downregulated by cAMP in normal, but not in SV40-transformed cells, suggesting that in normal cells epiregulin and amphiregulin activity is downregulated by a feedback mechanism that may be lost in SV40-transformed cells and their loss of downregulation may be involved in the development of ovarian tumors.