BACKGROUND: The links between the p53/MDM2 pathway and the expression of pro-oncogenic immune inhibitory receptors in tumor cells are undefined. In this report, we evaluate whether there is p53 and/or MDM2 dependence in the expression of two key immune receptors, CD276 and PD-L1. METHODS: Proximity ligation assays were used to quantify protein-protein interactions in situ in response to Nutlin-3. A panel of p53-null melanoma cells was created using CRISPR-Cas9 guide RNA mediated genetic ablation. Flow cytometric analyses were used to assess the impact of TP53 or ATG5 gene ablation, as well as the effects of Nutlin-3 and an ATM inhibitor on cell surface PD-L1 and CD276. Targeted siRNA was used to deplete CD276 to assess changes in cell cycle parameters by flow cytometry. A T-cell proliferation assay was used to assess activity of CD4+ T-cells as a function of ATG5 genotype. RESULTS: CD276 forms protein-protein interactions with MDM2 in response to Nutlin-3, similar to the known MDM2 interactors p53 and HSP70. Isogenic HCT116 p53-wt/null cancer cells demonstrated that CD276 is induced on the cell surface by Nutlin-3 in a p53-dependent manner. PD-L1 was also unexpectedly induced by Nutlin-3, but PD-L1 does not bind MDM2. The ATM inhibitor KU55993 reduced the levels of PD-L1 under conditions where Nutlin-3 induces PD-L1, indicating that MDM2 and ATM have opposing effects on PD-L1 steady-state levels. PD-L1 is also up-regulated in response to genetic ablation of TP53 in A375 melanoma cell clones under conditions in which CD276 remains unaffected. A549 cells with a deletion in the ATG5 gene up-regulated only PD-L1, further indicating that PD-L1 and CD276 are under distinct genetic control. CONCLUSION: Genetic inactivation of TP53, or the use of the MDM2 ligand Nutlin-3, alters the expression of the immune blockade receptors PD-L1 and CD276. The biological function of elevated CD276 is to promote altered cell cycle progression in response to Nutlin-3, whilst the major effect of elevated PD-L1 is T-cell suppression. These data indicate that TP53 gene status, ATM and MDM2 influence PD-L1 and CD276 paralogs on the cell surface. These data have implications for the use of drugs that target the p53 pathway as modifiers of immune checkpoint receptor expression.

• Klíčová slova
• Gene editing, MDM2, Nutlin-3, Protein-protein interactions, p53,
• MeSH
• antigeny B7 genetika   MeSH
• antigeny CD274 genetika   MeSH
• buněčný cyklus účinky léků   genetika   MeSH
• buňky A549 MeSH
• HCT116 buňky MeSH
• imidazoly farmakologie   MeSH
• lidé MeSH
• ligandy MeSH
• melanom farmakoterapie   MeSH
• nádorové buněčné linie MeSH
• nádorový supresorový protein p53 genetika   MeSH
• piperaziny farmakologie   MeSH
• proliferace buněk účinky léků   genetika   MeSH
• protoonkogenní proteiny c-mdm2 genetika   MeSH
• upregulace účinky léků   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny B7 MeSH
• antigeny CD274 MeSH
• CD274 protein, human MeSH Prohlížeč
• CD276 protein, human MeSH Prohlížeč
• imidazoly MeSH
• ligandy MeSH
• MDM2 protein, human MeSH Prohlížeč
• nádorový supresorový protein p53 MeSH
• nutlin 3 MeSH Prohlížeč
• piperaziny MeSH
• protoonkogenní proteiny c-mdm2 MeSH

Abnormal rates of growth together with metastatic potential and lack of susceptibility to cellular signals leading to apoptosis are widely investigated characteristics of tumors that develop via genetic or epigenetic mechanisms. Moreover, in the growing tumor, cells are exposed to insufficient nutrient supply, low oxygen availability (hypoxia) and/or reactive oxygen species. These physiological stresses force them to switch into more adaptable and aggressive phenotypes. This paper summarizes the role of two key mediators of cellular stress responses, namely p53 and HIF, which significantly affect cancer progression and compromise treatment outcomes. Furthermore, it describes cross-talk between these factors.

• MeSH
• faktor 1 indukovatelný hypoxií metabolismus   MeSH
• fyziologický stres MeSH
• hypoxie buňky MeSH
• karcinogeneze genetika   metabolismus   MeSH
• lidé MeSH
• nádorový supresorový protein p53 metabolismus   MeSH
• nádory genetika   metabolismus   MeSH
• poškození DNA * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• faktor 1 indukovatelný hypoxií MeSH
• nádorový supresorový protein p53 MeSH
• TP53 protein, human MeSH Prohlížeč

Hot spot mutant p53 (mutp53) proteins exert oncogenic gain-of-function activities. Binding of mutp53 to DNA is assumed to be involved in mutp53-mediated repression or activation of several mutp53 target genes. To investigate the importance of DNA topology on mutp53-DNA recognition in vitro and in cells, we analyzed the interaction of seven hot spot mutp53 proteins with topologically different DNA substrates (supercoiled, linear and relaxed) containing and/or lacking mutp53 binding sites (mutp53BS) using a variety of electrophoresis and immunoprecipitation based techniques. All seven hot spot mutp53 proteins (R175H, G245S, R248W, R249S, R273C, R273H and R282W) were found to have retained the ability of wild-type p53 to preferentially bind circular DNA at native negative superhelix density, while linear or relaxed circular DNA was a poor substrate. The preference of mutp53 proteins for supercoiled DNA (supercoil-selective binding) was further substantiated by competition experiments with linear DNA or relaxed DNA in vitro and ex vivo. Using chromatin immunoprecipitation, the preferential binding of mutp53 to a sc mutp53BS was detected also in cells. Furthermore, we have shown by luciferase reporter assay that the DNA topology influences p53 regulation of BAX and MSP/MST1 promoters. Possible modes of mutp53 binding to topologically constrained DNA substrates and their biological consequences are discussed.

• MeSH
• intracelulární signální peptidy a proteiny MeSH
• lidé MeSH
• mutace * MeSH
• mutantní proteiny chemie   genetika   metabolismus   MeSH
• nádorové buněčné linie MeSH
• nádorový supresorový protein p53 chemie   genetika   metabolismus   MeSH
• plazmidy genetika   MeSH
• promotorové oblasti (genetika) genetika   MeSH
• protein X asociovaný s bcl-2 genetika   MeSH
• protein-serin-threoninkinasy genetika   MeSH
• regulace genové exprese genetika   MeSH
• substrátová specifita MeSH
• superhelikální DNA chemie   metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• intracelulární signální peptidy a proteiny MeSH
• mutantní proteiny MeSH
• nádorový supresorový protein p53 MeSH
• protein X asociovaný s bcl-2 MeSH
• protein-serin-threoninkinasy MeSH
• STK4 protein, human MeSH Prohlížeč
• superhelikální DNA MeSH

Activation of the Hsp90 chaperone system is a characteristic of cancer cells. The regulation of chaperone activities involves their interaction with cochaperones; therefore we investigated the expression of Hsp70 and Hsp90 and their specific co-chaperones HOP and CHIP in cancer cell lines and primary cancers. Inhibition of Hsp90 by 17AAG increased the levels of Hsp70, Hsp90 and HOP but not CHIP mRNA in cancer cells. These changes are linked to activation of the HSF1 transcription factor and we show that the HOP promoter contains HSF1 binding sites, and that HSF1 binding to the HOP promoter is increased following 17AAG. The lack of alteration in the co-chaperone CHIP is explained by a lack of HSF response elements in the CHIP promoter. Non-proliferating cells expressed higher levels of CHIP and lower HOP, Hsp70 and Hsp90 levels compared to proliferating cells. Decreased expression of CHIP in proliferating cancer cells is in keeping with its proposed tumor suppressor properties, while over-expression of HOP in proliferating cells may contribute to excessive Hsp90 activity and stabilization of client proteins in tumors. In a panel of colorectal cancer samples, increased expression of Hsp70 and an increased ratio of HOP to CHIP were found, and were associated with decreased median survival. These data indicate that multiple changes occur in the chaperone/co-chaperone system in cancer that impact patient survival. It is likely that the ability to identify individual alterations to this system will be beneficial for treatment strategy decisions, particularly those that employ chaperone inhibitors.

• MeSH
• benzochinony farmakologie   MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• HCT116 buňky MeSH
• homeodoménové proteiny genetika   metabolismus   MeSH
• Kaplanův-Meierův odhad MeSH
• lidé MeSH
• makrocyklické laktamy farmakologie   MeSH
• nádorové supresorové proteiny genetika   metabolismus   MeSH
• nádory tračníku metabolismus   MeSH
• prognóza MeSH
• proliferace buněk účinky léků   MeSH
• promotorové oblasti (genetika) účinky léků   MeSH
• proteiny tepelného šoku HSP70 genetika   metabolismus   MeSH
• proteiny tepelného šoku HSP90 antagonisté a inhibitory   genetika   metabolismus   MeSH
• regulace genové exprese u nádorů účinky léků   MeSH
• responzivní elementy genetika   MeSH
• transkripční faktory tepelného šoku MeSH
• transkripční faktory genetika   metabolismus   MeSH
• ubikvitinligasy genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• benzochinony MeSH
• DNA vazebné proteiny MeSH
• homeodoménové proteiny MeSH
• HOPX protein, human MeSH Prohlížeč
• HSF1 protein, human MeSH Prohlížeč
• makrocyklické laktamy MeSH
• nádorové supresorové proteiny MeSH
• proteiny tepelného šoku HSP70 MeSH
• proteiny tepelného šoku HSP90 MeSH
• STUB1 protein, human MeSH Prohlížeč
• tanespimycin MeSH Prohlížeč
• transkripční faktory tepelného šoku MeSH
• transkripční faktory MeSH
• ubikvitinligasy MeSH

BACKGROUND: Cisplatin and its derivatives are commonly used anti-cancer drugs. However, cisplatin has clinical limitations including serious side effects and frequent emergence of intrinsic or acquired resistance. Thus, the novel platinum(IV) complex LA-12 represents a promising treatment modality, which shows increased intracellular penetration resulting in improved cytotoxicity in various cancer cell lines, including cisplatin resistant cells. RESULTS: LA-12 disrupts cellular proliferation regardless of the p53 status in the cells, however the potency of the drug is greatly enhanced by the presence of a functional p53, indicating several mechanisms of action. Similarly to cisplatin, an interaction of LA-12 with molecular chaperone Hsp90 was proposed. Binding of LA-12 to Hsp90 was demonstrated by Hsp90 immunoprecipitation followed by platinum measurement using atomic absorption spectrometry (AAS). An inhibitory effect of LA-12 on Hsp90 chaperoning function was shown by decrease of Hsp90-assisted wild-type p53 binding to p21WAF1 promoter sequence in vitro and by accelerated ubiqutination and degradation of primarily unfolded mutant p53 proteins in cells exposed to LA-12. CONCLUSIONS: To generalize our findings, LA-12 induced degradation of other Hsp90 client proteins such as Cyclin D1 and estrogen receptor was shown and proved as more efficient in comparison with cisplatin. This newly characterised molecular mechanism of action opens opportunities to design new cancer treatment strategy profitable from unique LA-12 properties, which combine DNA damaging and Hsp90 inhibitory effects.

• MeSH
• amantadin analogy a deriváty   farmakologie   MeSH
• cisplatina farmakologie   MeSH
• imunoprecipitace MeSH
• inhibitor p21 cyklin-dependentní kinasy účinky léků   metabolismus   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádorový supresorový protein p53 účinky léků   metabolismus   MeSH
• organoplatinové sloučeniny farmakologie   MeSH
• proteiny tepelného šoku HSP90 účinky léků   metabolismus   MeSH
• protinádorové látky farmakologie   MeSH
• spektrofotometrie atomová MeSH
• western blotting MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• amantadin MeSH
• bis(acetato)(1-adamantylamine)amminedichloroplatinum(IV) MeSH Prohlížeč
• CDKN1A protein, human MeSH Prohlížeč
• cisplatina MeSH
• inhibitor p21 cyklin-dependentní kinasy MeSH
• nádorový supresorový protein p53 MeSH
• organoplatinové sloučeniny MeSH
• proteiny tepelného šoku HSP90 MeSH
• protinádorové látky MeSH