TP73 is a member of the TP53 gene family and produces N- and C-terminal protein isoforms through alternative promoters, alternative translation initiation and alternative splicing. Most notably, p73 protein isoforms may either contain a p53-like transactivation domain (TAp73 isoforms) or lack this domain (ΔTAp73 isoforms) and these variants have opposing or independent functions. To date, there is a lack of well-characterised isoform-specific p73 antibodies. Here, we produced polyclonal and monoclonal antibodies to N-terminal p73 variants and the C-terminal p73α isoform, the most common variant in human tissues. These reagents show that TAp73 is a marker of multiciliated epithelial cells, while ΔTAp73 is a marker of non-proliferative basal/reserve cells in squamous epithelium. We were unable to detect ΔNp73 variant proteins, in keeping with recent data that this is a minor form in human tissues. Most cervical squamous cell carcinomas (79%) express p73α, and the distribution of staining in basal cells correlated with lower tumour grade. TAp73 was found in 17% of these tumours, with a random distribution and no association with clinicopathological features. These data indicate roles for ΔTAp73 in maintaining a non-proliferative state of undifferentiated squamous epithelial cells and for TAp73 in the production of differentiated multiciliated cells.

• MeSH
• Epithelial Cells metabolism   MeSH
• Humans MeSH
• Antibodies, Monoclonal MeSH
• Cell Line, Tumor MeSH
• Uterine Cervical Neoplasms metabolism   pathology   genetics   MeSH
• Neoplasms metabolism   pathology   genetics   MeSH
• Protein Isoforms * metabolism   genetics   MeSH
• Tumor Protein p73 * metabolism   genetics   MeSH
• Carcinoma, Squamous Cell metabolism   pathology   genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Nejnovější poznatky v biologii chronické lymfocytární leukemie (CLL) mají významný dopad na léčbu tohoto onemocnění. Buňky CLL vykazují závislost na zvýšené expresi BCL-2, autonomní BCR signalizaci a vyznačují se nadměrnou expresí delta izoformy p110 PI3K kinázy a BTK kinázy, která podporuje přežívání nádorových buněk. Cílená léčba monoklonálními protilátkami rituximabem a obinutuzumabem spolu s malými molekulami, jako je ibrutinib, idelalisib a venetoklax, výrazně rozšířila terapeutické možnosti, což vedlo ke zlepšení celkového přežití pacientů. V této souvislosti je pozoruhodné, že pacienti, kteří zahájili léčbu ibrutinibem, vykazují míru přežití srovnatelnou s celkovou populací. Ne všechny problémy spojené s CLL však byly vyřešeny, neboť stále přetrvávají otázky týkající se dysfunkce imunitního systému a sekundárních malignit, jednotnosti léčebných přístupů pro pacienty s vysokým a nízkým rizikem, dlouhodobých strategií pro mladé pacienty a terapie pro pacienty s Richterovou transformací. Přestože inhibitory BTK a BCL-2 mohou pozitivně ovlivnit imunitní systém, problémy spojené s infekcemi a sekundárními nádory přetrvávají. Pokud jde o Richterovu transformaci, identifikace specifických genetických abnormalit může v budoucnu umožnit cílenější a účinnější léčbu, včetně terapie CAR-T a bispecifických protilátek.

New findings in the biology of chronic lymphocytic leukemia (CLL) have major implications for the treatment of this disease. CLL cells exhibit a dependence on increased expression of BCL-2, an autonomous BCR signaling pathway, and are charac­terized by overexpression of the p110 PI3K delta kinase isoform and BTK kinase, which promotes tumor cell survival. Targeted therapies such as the monoclonal antibodies rituximab and obinutuzumab, along with small molecules such as ibrutinib, idelalisib, and venetoclax, have dramatically expanded therapeutic options, resulting in improved overall patient survival. In this context, it is note­worthy that patients starting treatment with ibrutinib have survival rates comparable to the general population. However, not all issues have been resolved, as there are questions regarding the immune system, consistency in treatment approaches and long-term strategies for young patients, especially those with Richter transformation. Although BTK and BCL-2 inhibitors can positively influence the immune system, we still face challenges related to infections and secondary tumors. Regarding Richter transformation, identification of specific genetic abnormalities may allow more targeted and effective therapies in the future, including CAR-T therapy and bispecific antibodies.

• Keywords
• Richterova transformace,
• MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell drug therapy   genetics   pathology   MeSH
• Immune System immunology   pathology   MeSH
• Humans MeSH
• Tumor Suppressor Protein p53 genetics   MeSH
• Agammaglobulinaemia Tyrosine Kinase antagonists & inhibitors   MeSH
• Antineoplastic Agents administration & dosage   therapeutic use   MeSH
• Antineoplastic Protocols MeSH
• Proto-Oncogene Proteins c-bcl-2 antagonists & inhibitors   MeSH
• Check Tag
• Humans MeSH

The p53 family of proteins evolved from a common ancestor into three separate genes encoding proteins that act as transcription factors with distinct cellular roles. Isoforms of each member that lack specific regions or domains are suggested to result from alternative transcription start sites, alternative splicing or alternative translation initiation, and have the potential to exponentially increase the functional repertoire of each gene. However, evidence supporting the presence of individual protein variants at functional levels is often limited and is inferred by mRNA detection using highly sensitive amplification techniques. We provide a critical appraisal of the current evidence for the origins, expression, functions and regulation of p53-family isoforms. We conclude that despite the wealth of publications, several putative isoforms remain poorly established. Future research with improved technical approaches and the generation of isoform-specific protein detection reagents is required to establish the physiological relevance of p53-family isoforms in health and disease. In addition, our analyses suggest that p53-family variants evolved partly through convergent rather than divergent evolution from the ancestral gene.

• MeSH
• Alternative Splicing * MeSH
• Humans MeSH
• RNA, Messenger metabolism   genetics   MeSH
• Evolution, Molecular MeSH
• Tumor Suppressor Protein p53 * metabolism   genetics   MeSH
• Transcription Initiation Site MeSH
• Protein Isoforms * genetics   metabolism   MeSH
• Gene Expression Regulation MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

Oncogene-induced replication stress has been recognized as a major cause of genome instability in cancer cells. Increased expression of cyclin E1 caused by amplification of the CCNE1 gene is a common cause of replication stress in various cancers. Protein phosphatase magnesium-dependent 1 delta (PPM1D) is a negative regulator of p53 and has been implicated in termination of the cell cycle checkpoint. Amplification of the PPM1D gene or frameshift mutations in its final exon promote tumorigenesis. Here, we show that PPM1D activity further increases the replication stress caused by overexpression of cyclin E1. In particular, we demonstrate that cells expressing a truncated mutant of PPM1D progress faster from G1 to S phase and fail to complete licensing of the replication origins. In addition, we show that transcription-replication collisions and replication fork slowing caused by CCNE1 overexpression are exaggerated in cells expressing the truncated PPM1D. Finally, replication speed and accumulation of focal DNA copy number alterations caused by induction of CCNE1 expression was rescued by pharmacological inhibition of PPM1D. We propose that increased activity of PPM1D suppresses the checkpoint function of p53 and thus promotes genome instability in cells expressing the CCNE1 oncogene.

• MeSH
• Cyclin E genetics   metabolism   MeSH
• Humans MeSH
• Tumor Suppressor Protein p53 * genetics   metabolism   MeSH
• Neoplasms * MeSH
• Genomic Instability MeSH
• Protein Phosphatase 2C genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Cell cycle checkpoints, oncogene-induced senescence and programmed cell death represent intrinsic barriers to tumorigenesis. Protein phosphatase magnesium-dependent 1 (PPM1D) is a negative regulator of the tumour suppressor p53 and has been implicated in termination of the DNA damage response. Here, we addressed the consequences of increased PPM1D activity resulting from the gain-of-function truncating mutations in exon 6 of the PPM1D. We show that while control cells permanently exit the cell cycle and reside in senescence in the presence of DNA damage caused by ionising radiation or replication stress induced by the active RAS oncogene, RPE1-hTERT and BJ-hTERT cells carrying the truncated PPM1D continue proliferation in the presence of DNA damage, form micronuclei and accumulate genomic rearrangements revealed by karyotyping. Further, we show that increased PPM1D activity promotes cell growth in the soft agar and formation of tumours in xenograft models. Finally, expression profiling of the transformed clones revealed dysregulation of several oncogenic and tumour suppressor pathways. Our data support the oncogenic potential of PPM1D in the context of exposure to ionising radiation and oncogene-induced replication stress.

• MeSH
• Cell Death genetics   MeSH
• Humans MeSH
• Mice MeSH
• Cell Transformation, Neoplastic * genetics   MeSH
• Tumor Suppressor Protein p53 genetics   metabolism   MeSH
• DNA Damage * genetics   MeSH
• Cell Proliferation genetics   MeSH
• Protein Phosphatase 2C * genetics   metabolism   MeSH
• Phosphoprotein Phosphatases genetics   metabolism   MeSH
• Cellular Senescence * genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

As species adapt to climatic changes, temperature-dependent functions of p53 in development, metabolism and cancer will adapt as well. Structural analyses of p53 epitopes interacting in response to environmental stressors, such as heat, may uncover physiologically relevant functions of p53 in cell regulation and genomic adaptations. Here we explore the multiple p53 elephant paradigm with an experimentally validated in silico model showing that under heat stress some p53 copies escape negative regulation by the MDM2 E3 ubiquitin ligase. Multiple p53 isoforms have evolved naturally in the elephant thus presenting a unique experimental system to study the scope of p53 functions and the contribution of environmental stressors to DNA damage. We assert that fundamental insights derived from studies of a historically heat-challenged mammal will provide important insights directly relevant to human biology in the light of climate change when 'heat' may introduce novel challenges to our bodies and health.

• Publication type
• Journal Article MeSH
• Review MeSH

Anthracyclines, such as doxorubicin (adriamycin), daunorubicin, or epirubicin, rank among the most effective agents in classical anticancer chemotherapy. However, cardiotoxicity remains the main limitation of their clinical use. Topoisomerase IIβ has recently been identified as a plausible target of anthracyclines in cardiomyocytes. We examined the putative topoisomerase IIβ selective agent XK469 as a potential cardioprotective and designed several new analogs. In our experiments, XK469 inhibited both topoisomerase isoforms (α and β) and did not induce topoisomerase II covalent complexes in isolated cardiomyocytes and HL-60, but induced proteasomal degradation of topoisomerase II in these cell types. The cardioprotective potential of XK469 was studied on rat neonatal cardiomyocytes, where dexrazoxane (ICRF-187), the only clinically approved cardioprotective, was effective. Initially, XK469 prevented daunorubicin-induced toxicity and p53 phosphorylation in cardiomyocytes. However, it only partially prevented the phosphorylation of H2AX and did not affect DNA damage measured by Comet Assay. It also did not compromise the daunorubicin antiproliferative effect in HL-60 leukemic cells. When administered to rabbits to evaluate its cardioprotective potential in vivo, XK469 failed to prevent the daunorubicin-induced cardiac toxicity in either acute or chronic settings. In the following in vitro analysis, we found that prolonged and continuous exposure of rat neonatal cardiomyocytes to XK469 led to significant toxicity. In conclusion, this study provides important evidence on the effects of XK469 and its combination with daunorubicin in clinically relevant doses in cardiomyocytes. Despite its promising characteristics, long-term treatments and in vivo experiments have not confirmed its cardioprotective potential.

• MeSH
• Anthracyclines * toxicity   therapeutic use   MeSH
• Quinoxalines * MeSH
• Daunorubicin toxicity   therapeutic use   MeSH
• DNA Topoisomerases, Type II metabolism   therapeutic use   MeSH
• Doxorubicin toxicity   MeSH
• Topoisomerase II Inhibitors * toxicity   therapeutic use   MeSH
• Cardiotoxicity MeSH
• Rabbits MeSH
• Rats MeSH
• DNA Damage MeSH
• Antibiotics, Antineoplastic toxicity   MeSH
• Animals MeSH
• Check Tag
• Rabbits MeSH
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

The p53 protein is a key tumor suppressor and the most commonly mutated and down-regulated protein in human tumors. It functions mainly through interaction with DNA, and p53 acts as a transcription factor that recognizes the so-called p53 target sites on the promoters of various genes. P53 has been shown to exist as many isoforms, including three C-terminal isoforms that are produced by alternative splicing. Because the C-terminal domain is responsible for sequence-nonspecific binding and regulation of p53 binding, we have analyzed DNA recognition by these C-terminal isoforms. Using atomic force microscopy, we show for the first time that all C-terminal isoforms recognize superhelical DNA. It is particularly noteworthy that a sequence-specific p53 consensus binding site is bound by p53α and β isoforms with similar affinities, whilst p53α shows higher binding to a quadruplex sequence than both p53β and p53γ, and p53γ loses preferential binding to both the consensus binding sequence and the quadruplex-forming sequence. These results show the important role of the variable p53 C-terminal amino acid sequences for DNA recognition.

• MeSH
• Alternative Splicing * MeSH
• DNA genetics   metabolism   MeSH
• Humans MeSH
• Tumor Suppressor Protein p53 * metabolism   MeSH
• Protein Isoforms genetics   metabolism   MeSH
• Base Sequence MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Cellular stress conditions activate p53-dependent pathways to counteract the inflicted damage. To achieve the required functional diversity, p53 is subjected to numerous post-translational modifications and the expression of isoforms. Little is yet known how p53 has evolved to respond to different stress pathways. The p53 isoform p53/47 (p47 or ΔNp53) is linked to aging and neural degeneration and is expressed in human cells via an alternative cap-independent translation initiation from the 2nd in-frame AUG at codon 40 (+118) during endoplasmic reticulum (ER) stress. Despite an AUG codon in the same location, the mouse p53 mRNA does not express the corresponding isoform in either human or mouse-derived cells. High-throughput in-cell RNA structure probing shows that p47 expression is attributed to PERK kinase-dependent structural alterations in the human p53 mRNA, independently of eIF2α. These structural changes do not take place in murine p53 mRNA. Surprisingly, PERK response elements required for the p47 expression are located downstream of the 2nd AUG. The data show that the human p53 mRNA has evolved to respond to PERK-mediated regulation of mRNA structures in order to control p47 expression. The findings highlight how p53 mRNA co-evolved with the function of the encoded protein to specify p53-activities under different cellular conditions.

• MeSH
• eIF-2 Kinase genetics   metabolism   MeSH
• Humans MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Mice MeSH
• Tumor Suppressor Protein p53 * genetics   metabolism   MeSH
• Protein Processing, Post-Translational MeSH
• Protein Isoforms metabolism   MeSH
• Endoplasmic Reticulum Stress * genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The TP63 gene encodes two major protein variants; TAp63 contains a p53-like transcription domain and consequently has tumor suppressor activities whereas ΔNp63 lacks this domain and acts as an oncogene. The two variants show distinct expression patterns in normal tissues and tumors, with lymphocytes and lymphomas/leukemias expressing TAp63, and basal epithelial cells and some carcinomas expressing high levels of ΔNp63, most notably squamous cell carcinomas (SCC). Whilst the transcriptional functions of TAp63 and ΔNp63 isoforms are known, the mechanisms involved in their regulation are poorly understood. Using squamous epithelial cells that contain high levels of ΔNp63 and low/undetectable TAp63, the DNA demethylating agent decitabine (5-aza-2'-deoxycytidine, 5-dAza) caused a dose-dependent increase in TAp63, with a simultaneous reduction in ΔNp63, indicating DNA methylation-dependent regulation at the isoform-specific promoters. The basal cytokeratin KRT5, a direct ΔNp63 transcriptional target, was also reduced, confirming functional alteration of p63 activity after DNA demethylation. We also showed high level methylation of three CpG sites in the TAP63 promoter in these cells, which was reduced by decitabine. DNMT1 depletion using inducible shRNAs partially replicated these effects, including an increase in the ratio of TAP63:ΔNP63 mRNAs, a reduction in ΔNp63 protein and reduced KRT5 mRNA levels. Finally, high DNA methylation levels were found at the TAP63 promoter in clinical SCC samples and matched normal tissues. We conclude that DNA methylation at the TAP63 promoter normally silences transcription in squamous epithelial cells, indicating DNA methylation as a therapeutic approach to induce this tumor suppressor in cancer. That decitabine simultaneously reduced the oncogenic activity of ΔNp63 provides a "double whammy" for SCC and other p63-positive carcinomas. Whilst a variety of mechanisms may be involved in producing the opposite effects of DNA demethylation on TAp63 and ΔNp63, we propose an "either or" mechanism in which TAP63 transcription physically interferes with the ability to initiate transcription from the downstream ΔNP63 promoter on the same DNA strand. This mechanism can explain the observed inverse expression of p63 isoforms in normal cells and cancer.

• Publication type
• Journal Article MeSH