BackgroundReactivation of p53 by either gene transfer or pharmacologic approaches may compensate for loss of p19Arf or excess mdm2 expression, common events in melanoma and glioma. In our previous work, we constructed the pCLPG retroviral vector where transgene expression is controlled by p53 through a p53-responsive promoter. The use of this vector to introduce p19Arf into tumor cells that harbor p53wt should yield viral expression of p19Arf which, in turn, would activate the endogenous p53 and result in enhanced vector expression and tumor suppression. Since nutlin-3 can activate p53 by blocking its interaction with mdm2, we explored the possibility that the combination of p19Arf gene transfer and nutlin-3 drug treatment may provide an additive benefit in stimulating p53 function.MethodsB16 (mouse melanoma) and C6 (rat glioma) cell lines, which harbor p53wt, were transduced with pCLPGp19 and these were additionally treated with nutlin-3 or the DNA damaging agent, doxorubicin. Viral expression was confirmed by Western, Northern and immunofluorescence assays. p53 function was assessed by reporter gene activity provided by a p53-responsive construct. Alterations in proliferation and viability were measured by colony formation, growth curve, cell cycle and MTT assays. In an animal model, B16 cells were treated with the pCLPGp19 virus and/or drugs before subcutaneous injection in C57BL/6 mice, observation of tumor progression and histopathologic analyses.ResultsHere we show that the functional activation of endogenous p53wt in B16 was particularly challenging, but accomplished when combined gene transfer and drug treatments were applied, resulting in increased transactivation by p53, marked cell cycle alteration and reduced viability in culture. In an animal model, B16 cells treated with both p19Arf and nutlin-3 yielded increased necrosis and decreased BrdU marking. In comparison, C6 cells were quite susceptible to either treatment, yet p53 was further activated by the combination of p19Arf and nutlin-3.ConclusionsTo the best of our knowledge, this is the first study to apply both p19Arf and nutlin-3 for the stimulation of p53 activity. These results support the notion that a p53 responsive vector may prove to be an interesting gene transfer tool, especially when combined with p53-activating agents, for the treatment of tumors that retain wild-type p53.

Humans have been using fire for hundreds of millennia, creating an ancestral expansion toward the nocturnal niche. The new adaptive challenges faced at night were recurrent enough to amplify existing psychological variation in our species. Night-time is dangerous and mysterious, so it selects for individuals with higher tendencies for paranoia, risk-taking, and sociability (because of security in numbers). During night-time, individuals are generally tired and show decreased self-control and increased impulsive behaviors. The lower visibility during night-time favors the partial concealment of identity and opens more opportunities for disinhibition of self-interested behaviors. Indeed, individuals with an evening-oriented chronotype are more paranoid, risk-taking, extraverted, impulsive, promiscuous, and have higher antisocial personality traits. However, under some circumstances, such as respiratory pandemics, the psychobehavioral traits favored by the nocturnal niche might be counter-productive, increasing contagion rates of a disease that can evade the behavioral immune system because its disease cues are often nonexistent or mild. The eveningness epidemiological liability hypothesis presented here suggests that during the COVID-19 pandemic, the evening-oriented psychobehavioral profile can have collectively harmful consequences: there is a clash of core tendencies between the nocturnal chronotype and the recent viral transmission-mitigating safety guidelines and rules. The pandemic safety protocols disrupt much normal social activity, particularly at night when making new social contacts is desired. The SARS-CoV-2 virus is contagious even in presymptomatic and asymptomatic individuals, which enables it to mostly evade our evolved contagious disease avoidance mechanisms. A growing body of research has indirectly shown that individual traits interfering with social distancing and anti-contagion measures are related to those of the nocturnal chronotype. Indeed, some of the social contexts that have been identified as superspreading events occur at night, such as in restaurants, bars, and nightclubs. Furthermore, nocturnal environmental conditions favor the survival of the SARS-CoV-2 virus much longer than daytime conditions. We compare the eveningness epidemiological liability hypothesis with other factors related to non-compliance with pandemic safety protocols, namely sex, age, and life history. Although there is not yet a direct link between the nocturnal chronotype and non-compliance with pandemic safety protocols, security measures and future empirical research should take this crucial evolutionary mismatch and adaptive metaproblem into account, and focus on how to avoid nocturnal individuals becoming superspreaders, offering secure alternatives for nocturnal social activities.

Alternative treatments for cancer using gene therapy approaches have shown promising results and some have even reached the marketplace. Even so, additional improvements are needed, such as employing a strategically chosen promoter to drive expression of the transgene in the target cell. Previously, we described viral vectors where high-level transgene expression was achieved using a p53-responsive promoter. Here we present an adenoviral vector (AdPGp53) where p53 is employed to regulate its own expression and which outperforms a traditional vector when tested in a model of gene therapy for prostate cancer. The functionality of AdPGp53 and AdCMVp53 were compared in human prostate carcinoma cell lines. AdPGp53 conferred greatly enhanced levels of p53 protein and induction of the p53 target gene, p21, as well as superior cell killing by a mechanism consistent with apoptosis. DU145 cells were susceptible to induction of death with AdPGp53, yet PC3 cells were quite resistant. Though AdCMVp53 was shown to be reliable, extremely high-level expression of p53 offered by AdPGp53 was necessary for tumor suppressor activity in PC3 and DU145. In situ gene therapy experiments revealed tumor inhibition and increased overall survival in response to AdPGp53, but not AdCMVp53. Upon histologic examination, only AdPGp53 treatment was correlated with the detection of both p53 and TUNEL-positive cells. This study points to the importance of improved vector performance for gene therapy of prostate cancer.

Título da Tese: Construção e caracterização de vetor adenoviral com promotor responsívo ao seu próprio trangene p53 e sua comparação com um vetor de promoção constitutiva. .